hTERT gene knockdown enhances response to radio- and chemotherapy in head and neck cancer cell lines through a DNA damage pathway modification

The aim of the study was to analyze the effect of hTERT gene knockdown in HNSCC cells by using novel in vitro models of head and neck cancer (HNSCC), as well as improving its personalized therapy. To obtain the most efficient knockdown siRNA, shRNA-bearing lentiviral vectors were used. The efficiency of hTERT silencing was verified with qPCR, Western blot, and immunofluorescence staining. Subsequently, the type of cell death and DNA repair mechanism induction after hTERT knockdown was assessed with the same methods, followed by flow cytometry. The effect of a combined treatment with hTERT gene knockdown on Double-Strand Breaks levels was also evaluated by flow cytometry. Results showed that the designed siRNAs and shRNAs were effective in hTERT knockdown in HNSCC cells. Depending on a cell line, hTERT knockdown led to a cell cycle arrest either in phase G1 or phase S/G2. Induction of apoptosis after hTERT downregulation with siRNA was observed. Additionally, hTERT targeting with lentiviruses, followed by cytostatics administration, led to induction of apoptosis. Interestingly, an increase in Double-Strand Breaks accompanied by activation of the main DNA repair mechanism, NER, was also observed. Altogether, we conclude that hTERT knockdown significantly contributes to the efficacy of HNSCC treatment.

SCiENTiFiC REpoRTS | (2018) 8:5949 | DOI: 10.1038/s41598-018-24503-y adenoviral vectors) [3][4][5] . But due to the complexity of the process, there is still much to discover. Even if various mechanisms of cell death-including autophagy, mitotic catastrophe, and necrosis-share some common areas, it is still difficult to apply this knowledge to cancer therapy. Even targeting telomerase may appear less efficient than expected since some cancer cells can develop a telomerase-independent way of telomere restoration, i.e., Alternative Telomere Lengthening (ALT) 6 . Consequently, it is difficult to describe the associations between telomerase and cancer cell metabolism. In any case, it is difficult to transfer this knowledge into clinics.
RNA interference as an effective system for silencing gene expression has found its application in gene therapy. Given the transfection efficiency and ease of delivery, the use of siRNA is more advantageous than shRNA. Takahashi et al. (2009) observed higher siRNA transfection efficiency when compared to shRNA in cells with low proliferative potential 7 . Additionally, the lower molecular weight of siRNA-when compared to shRNA-makes these molecules easier to deliver to cells and viral and non-viral systems are the main methods of delivering interfering RNA particles into cells. Retroviral vectors including lentiviruses, adenoviruses, and adeno-associated viruses are especially considered as being potentially effective vectors in cancer gene therapy. However, the construction of a safe, efficient, and universal system is still a challenge [8][9][10][11] .
In this project we analyzed the efficiency and potency of RNA interference (siRNA and shRNA) directed against hTERT in order to eliminate cancer cells. And due to the complex nature of carcinogenesis processes and the contribution of many factors to the control of tumor growth, the possibility of using shRNA against telomerase may very well provide a novel approach when administrating cytostatics and/or radiation therapy. Consequently, it might lead to a reduction in drug and radiation doses, and a decrease in harmful side effects.

Results
Assessment of hTERT downregulation efficiency. Cells were subjected to an analysis of hTERT downregulation effect 72 hours after transfection. At that time a significant effect was observed in both FaDu and H103 cells at the transcriptional level (downregulation at 72%, p = 0.0003 and 69%, p ≤ 0.0001, relative to mock siRNA, respectively) as well as at the protein level (downregulation at 66%, p = 0.0003 and 94%, p ≤ 0.0001, respectively) when immunodetection was applied. The effect was less efficient but still significant after the next 4 days (altogether 7 days) in both cell lines at the transcriptional level (61%, p = 0.0012 and 66%, p ≤ 0.0001, respectively), as well as at the protein level (22%, p = 0.0328 and 63%, p = 0.0004, respectively) ( Fig. 1A,B). hTERT knockdown was even more persistent when shRNA was applied, showing a significant effect after 10 days of lentiviral infection in both FaDu and H103 cells at the transcriptional level (downregulation at 71%; p = 0.0106 and 64%; p = 0.0135, respectively) (qPCR) and protein level (downregulation at 84%; p = 0.0005 and H103 − 77%; p ≤ 0.0001, respectively) (immunofluorescence staining) (Fig. 1A,C).
Noteworthy, a significant telomere attrition was detected (qPCR) in the FaDu cell line after hTERT knockdown was conducted via siRNA (59%, p = 0.0134) and shRNA (79%, p = 0.0083) when compared to control samples. Interestingly, no telomere length alteration was observed in H103 cells (Fig. 1D). Furthermore, a significant depletion of a proliferation rate was observed in FaDu and H103 cells (Fig. 1E) when a microscopic observation was applied. Moreover, cell migration ability decreased significantly after hTERT knockdown with siRNA (wound healing assay). At different time intervals in both FaDu (p = 0.037 for 24 hours and 48 hours, p = 0.015 and p = 0.009 for 72 hours and 96 hours respectively) and H103 (p = 0.036 for 24 hours and p = 0.046 for 48 hours, respectively) migration potential decreased when assessed with a wound healing test (Fig. 1F).
Cell cycle arrest analysis. In order to analyze the impact of hTERT downregulation on the cell cycle, a flow cytometry analysis using propidium iodide was performed. In both cell lines, a significant increase in the apoptotic cell fraction was observed when compared to the control sample. In the FaDu cell line, on day 7, a fraction of the apoptotic cells reached 75.8%. In the control, however, it was barely detectable (3.23%). In H103 cells, distribution of fractions was not that vivid; apoptotic cells reached 47.9% compared to control (2.68%). Three days after transfection in the H103 cells, apoptotic fraction was increased to 22.9%. At the same time point in the FaDu cell line, elevated G1 fraction (from 46.6% to 62.2%) with simultaneous depletion of G2 fraction (from 28.1% to 13.1%) was observed, indicating a cell cycle arrest in G1 phase ( Fig. 2A). In case of hTERT gene silencing with shRNA, an increased percentage of G1 phase (10.3% more cells compared to control) and a decreased percentage of cells in G2 (13.6%) was also noticed. In H103 cells, an increase in the fraction of cells in G2 phase (11.4%) and a decrease in the G1 phase (19.2%) has been shown ( Fig. 2A).
Cell death assessment -flow cytometry analysis. Due to the demonstrated effect of hTERT gene silencing on cell cycle arrest and the ambiguous literature data suggesting the effect of hTERT gene silencing on apoptosis induction), an attempt to verify this theory was made. In both FaDu and H103 cell lines, an increased level of apoptosis markers was observed: in FaDu cells upregulation of CASP3, CASP9, and ANXA5 (179% p = 0.0012; -323% p ≤ 0.0001; -191% p = 0.001, respectively) at day 7. Also, in H103 cells, an elevated level of apoptosis markers was noticed 3 days after transfection (CASP3 − 158%, p = 0.0143; CASP9 -144%, p = 0.0119) when qPCR and Western blot analyzes were performed. The effect was most significant 7 days after transfection. hTERT gene expression at the transcriptional level measured by qPCR; (B) hTERT gene expression at the protein level (siRNA) with Western blot analysis. Semi-quantitative analysis of Western blot results using ImageJ software; (C) hTERT gene expression at the protein level (shRNA) by immunofluorescence staining, objective magnification 20x; (D) relative telomere length analysis by qPCR; (E) cell proliferation microscopic observation, objective magnification 20x; (F) cell migration wound healing assay, objective magnification 20x. CTRL -control cells transfected with non-specific siRNA/cells transduced with control lentiviral vector; siTERT -cells transfected with hTERT gene-targeted siRNA on day 3 rd and 7 th ; shTERT -cells transduced with lentiviral vectors containing shRNA. Data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 with comparisons indicated by lines. Moreover, on day 7 the increased expression of BECN1 was observed in H103 cells (BECN1 -143%, p = 0.0336) (Fig. 3A,B). Activation of apoptosis after hTERT knockdown was confirmed by flow cytometry with Annexin V and propidium iodide staining. An elevated fraction of apoptotic cells was demonstrated 7 days after transfection in cell line FaDu (from 0.27% to 35.7%; p < 0.0001) and cell line H103 (from 1.58% to 8.69%; p < 0.0002). In the case of FaDu cells, propidium iodide-positive necrotic fraction on the 7 th day was also observed (from 0.77% to 34.8%; p < 0.0001) (Fig. 3C).
Importantly, knockdown of hTERT via lentiviral vector did not cause activation of apoptosis as observed in the experiment with FaDu and H103 cell lines. However, an increased level of BECN1 was observed in H103 cells (229%, p ≤ 0.0001, relative to control) (Fig. 3D,E).

Induction of apoptosis after hTERT knockdown with cytostatics co-administration.
Studied cells were more susceptible to death when cytostatic compounds were combined with hTERT downregulation. In FaDu cells, however, the expression of BECN1-measured with qPCR at the transcription level-was elevated following docetaxel administration at 0.5 TPL, i.e., 275 nM (p = 0.0193) and 1 TPL, i.e., 550 nM (p = 0.0182) concentrations. CASP3 gene expression at 1 TPL of cisplatin (p = 0.0341) and docetaxel at 0.5 TPL (p = 0.0418) and 1 TPL (p = 0.0282) was also increased (Fig. 4A). A significant effect of hTERT downregulation and cytostatics administration on gene expression analyzed at the protein level was also noted. We observed an increase in BECN1 accumulation (IF assay) after administering cisplatin and docetaxel at a concentration of 1 TPL (p = 0.0009; p ≤ 0.0001 respectively) (Fig. 4B). A significant increase in the CASP3 level was also revealed after cisplatin administration (0.5 TPL [p = 0.0011] and 1 TPL [p ≤ 0.0001]) (Fig. 4C).
In H103 cells, expression of BECN1 at the transcription level was elevated after cisplatin was administered at doses of 1 TPL, i.e. 6.67 µM (p = 0.0372) and docetaxel at 0.5 TPL (p = 0.0098). There was an increase in gene expression of CASP3 after administering cisplatin at a concentration of 1 TPL (p = 0.0443) (Fig. 5A). Changes in expression of the BECN1 and CASP3 genes were also observed at the protein level. An increased expression of the BECN1 gene after cisplatin   siRNA -Analysis of apoptosis-and autophagy-related genes expression at the protein level by Western blot analysis. Semi-quantitative analysis of Western blot results using ImageJ software; (C) Apoptosis analysis by flow cytometry; (D) shRNA -Analysis of apoptosis-and autophagy-related genes expression at the transcriptional level with qPCR; (E) shRNA -Analysis of apoptosis-and autophagy-related genes expression at the protein level by immunofluorescence staining, objective magnification 20x. CTRL -control cells transfected with nonspecific siRNA/cells transduced with control lentiviral vector; siTERT -cells transfected with hTERT genetargeted siRNA on day 3 rd and 7 th ; shTERT -cells transduced with lentiviral vectors containing shRNA on day 10 th . Data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 with comparisons indicated by lines. shTERT d10/d10 -cells transduced with lentiviral vectors containing shRNA on day 10 th . Data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 with comparisons indicated by lines. Furthermore, measurement of the γH2AX level in hTERT knockdown cells exposed to ionizing radiation and cytostatics (cisplatin and docetaxel [0.5 and 1 TPL]) was performed for shRNA variant. In FaDu cells, an increased level of DSBs was demonstrated after administrating 0.5 TPL cisplatin alone without irradiation (0 Gy − 1.87 MFI, p = 0.0022) and with a dose of 0.5 Gy (1.40 MFI, p = 0.0158) and 2 Gy (1.59 MFI, p = 0.0055). In case of H103 cells, elevated levels of DSBs were noticed after the addition of 0.5 TPL cisplatin without irradiation (2.45 MFI, p = 0.0007) and radiation exposure of 0.5 Gy (1.8 MFI, p = 0.0026) and 1 Gy (2.09 MFI, p = 0.0012). Moreover, we observed an elevated DSBs level after 1 TPL cisplatin administration with 0.5 Gy (0.54 MFI, p = 0.0022) and without irradiation (0.74 MFI, p = 0.0244) (Fig. 7A).
Analysis of the protein level showed an increase in expression of the CSB gene (331%, p = 0.0003) (NER) in H103 cells at day 7 after transfection with siRNA. We also observed an elevated expression of the XRCC4 gene (224%, p = 0.0009) (NHEJ mechanism) on day 7. No increase in gene expression was detected concerning markers of the remaining DNA damage repair mechanisms (Fig. 8C).

Discussion
The primary aim of the study was to analyze the effect of hTERT gene knockdown on cancer cell metabolism in order to develop a personalized therapy of head and neck squamous cell carcinoma. The first part of the study focused on an efficient hTERT gene expression silencing. To obtain the most effective knockdown, cells were transfected with siRNA or transduced with shRNA-bearing lentiviral vectors. Subsequently, the types of cell death and DNA repair mechanisms activated after hTERT knockdown were assessed. The ability of establishing an HNSCC cell line after hTERT gene knockdown in order to create a model for assessing the effect of a combined treatment (chemotherapy and/or radiotherapy) was also evaluated.
The use of RNA interference (shRNA or siRNA) as a tool for hTERT gene silencing has been used in several types of cancer [12][13][14][15][16][17] . In the case of head and neck cancer, only a few studies have shown a significant therapeutic potential of hTERT silencing using RNA interference, thus revealing its impact on cell death and growth 18,19 . There is also a limited number of studies where lentiviral vectors were used as a tool to carry shRNA for stable silencing telomerase expression in head and neck cancer. When Yao et al. (2011) investigated the effect of hTERT gene silencing by shRNA in a murine model of nasopharyngeal cancer, they demonstrated its inhibitory result on cell growth. Moreover, the influence of hTERT gene knockdown on proliferation, migration, and cell invasion inhibition was observed 20 . Also, the effectiveness of the lentiviral vectors carrying shRNA for hTERT gene expression depletion in a KB cell line, apoptosis activation and cell cycle arrest lead to the inhibition of cell proliferation, as demonstrated by Chen et al. 21 .
In this paper, silencing of hTERT gene encoding the telomerase catalytic subunit in HNSCC cells using siR-NAs and shRNAs was performed. In the case of shRNA, lentiviral vectors were used as a carrier. For this purpose, a new shRNA molecule directed against the hTERT gene was designed and cloned into lentiviral vector pLV-THEM-GP1. A lentivector obtained this way (pLV-THEM-shTERT) has a strong H1 promoter that ensures high and stable shRNA expression.
In order to verify the functionality and efficiency of the hTERT silencing system, wide range tests were performed in FaDu and H103 established HNSCC cell lines. The analysis was made on day 3 and 7 after siRNA transfection, and on day 10 after transduction with lentiviral vectors.
The studies showed a significant reduction in hTERT gene expression at the transcriptional and protein levels in both cell lines using lentiviruses and siRNA. However, in the case of siRNA transfected cells, gene expression was restored to original levels on day 7 of the experiment. In order to confirm the efficiency of the designed sequence and to reveal the loss of telomerase catalytic function, the telomere length was measured. Both studied methods (siRNA/shRNA) successfully led to a significant shortening of telomeres in FaDu cell line. Moreover, in the case of the transfection with siRNA, this effect was observed as early as 3 days after the initial treatment. On the other hand, telomeres in H103 cell line were fully maintained after treatment with either lentiviruses or siRNA molecules, which may indicate the activation of an alternative lengthening of telomeres mechanism. The confirmation of proper functioning siRNA and shRNA was also the proliferation and migration inhibition of hTERT knockdown cells. Obtained results demonstrated the functionality of designed constructs (siRNA/shRNA).
The literature data thoroughly describing the effect of hTERT gene silencing using either siRNA or lentiviral vectors as shRNA carriers for head and neck cancer are limited, and do not indicate ambiguous molecular effects of the hTERT gene knockdown. Therefore, we decided to assess the type of cell death and DNA repair mechanisms activated following hTERT gene silencing in HNSCC cells. Furthermore, this study focuses on hTERT's knockdown ability to alter the HNSCC cell lines response to combined treatment (chemotherapy and/ or radiotherapy).
Presented literature data concerning different types of cancer indicate the activation of apoptosis after hTERT gene silencing achieved by either siRNA or shRNA. As far as head and neck cancer is concerned, we still lack data that support such a notion. Zhou et al. (2006) demonstrated the activation of apoptosis and the inhibition of tumor growth after hTERT knockdown using shRNA in laryngeal cancer murine model 22  To evaluate the effect of hTERT knockdown using the novel in vitro head and neck cancer model, cell death mechanism and cell cycle analysis were performed.
Due to the limited number and inconsistent literature data, we further studied the degree of apoptosis activation following the hTERT gene silencing and use of standard chemotherapeutics of head and neck cancer treatment (cisplatin and docetaxel). The analysis of gene expression-which are markers for these mechanisms-was carried out. In the case of apoptosis, expression levels of CASP3, CASP9, and ANXA5 genes were evaluated, whereas measurement of BECN1 expression was conducted as an autophagy-related gene.
When silencing the hTERT gene with siRNA, a significant increase in expression of the apoptosis markers CASP3, CASP9, and ANXA5 was shown at the transcriptional level on day 7. However, no changes were noted on day 3 except for the CASP9 gene. Decrease in BECN1 gene expression on days 3 and 7 at both the transcriptional and protein levels was also observed. In the H103 cell line, gene expression of CASP3 and CASP9 on day 3 and CASP9 gene on day 7 was observed to have magnified apoptosis induction. An increase in accumulation of the BECN1 gene at the protein level on day 7 was also demonstrated. In order to confirm the results indicating apoptosis activation, flow cytometric analysis was performed. An increase in late-stage apoptotic cells in both FaDu and H103 cell lines on day 7 was noticed. Moreover, in FaDu cells, the appearance of the necrotic fraction was observed. These results suggest that the silencing of hTERT gene expression using siRNA leads directly to the induction of apoptosis. When lentiviral vectors are used, we examined not only activation of apoptotic mechanism after the hTERT gene silencing alone, but also with the concomitant administration of cytostatics. This analysis was not possible to carry out with transfection of siRNA due to the high cell mortality. The results obtained in this study showed no activation of apoptosis after hTERT gene silencing using lentiviral vectors. Nevertheless, for the first time the effects of decreased expression of this gene on cell sensitivity to cisplatin and docetaxel on an in vitro HNSCC model were observed, as well as the induction of this cell death mechanism.
The activation of the apoptosis following hTERT knockdown suggests possible cell cycle arrest. The literature data do not clearly indicate the effect of this gene silencing on cell cycle in cancer cells. Zhong et al. (2010) showed an increase in G0/G1 cell fraction and a decrease in S/G2 phase in pancreatic cancer model 17 . Similar results were obtained by Xu et al. (2015) when examining the effect of hTERT gene silencing in a cellular model of liver cancer, as well as by Shi et al. (2014) in studies on cervical cancer 13,15 . Luo et al. (2009), investigating the effect of transfection with a plasmid encoding shRNA against the hTERT gene on ovarian cancer cells, showed that silencing of this gene results in cell cycle arrest in S-phase 31 .
In this paper, an increase in the percentage of FaDu cells in G1 phase and a decrease in the fraction of cells in G2 phase were noticed subsequent to siRNA/shRNA mediated knockdown. In the case of the H103, an increase in the fraction of cells in the S and G2 phases along with a decrease in the G1 phase was shown. These results indicate that, depending on the cell line, reduced hTERT expression results in cell cycle arrest either in phase G1 or S/ G2. Regardless, where the cell cycle is stopped is where all cells are ultimately directed onto the apoptosis pathway.
Moreover, the cell cycle analysis by flow cytometry allowed for additional confirmation of the induction of apoptosis following hTERT gene silencing (siRNA). In both cell lines, a significant increase in apoptotic sub-G1 cell fraction compared with control was observed. Shammas et al. (2005) in esophageal cancer and Dong et al. (2009) in the breast cancer model reported a similar increase in apoptotic cell fraction 27,32 . Gandellini et al. (2007) in the study of the effect of hTERT gene silencing on the induction of apoptosis in prostate cancer cells observed only a slight increase in this fraction 16 . Such an effect was not reported for pancreatic cancer 17 .
According to literature data, production of DSBs is directly connected not only with cell cycle arrest but also as a result of exposure to ionizing radiation 33 .
We demonstrated that hTERT knockdown (with shRNA) in both studied 34 cell lines resulted in an increase of DSBs compared to control. Such an effect was also detected after irradiation with 0.5 Gy and 1 Gy doses in FaDu cells. Increased sensitivity to ionizing radiation with dose of 2 Gy in H103 hTERT gene knockdown cells was demonstrated. Measurement of the level of DSBs following irradiation of cells with silenced hTERT gene with simultaneous administration of cytostatics (cisplatin and docetaxel) was also performed. After the administration of cisplatin and exposure to radiation (0.5 TPL and dose of 0.5 Gy and 1 Gy, and 1 TPL and dose of 2 Gy), an increase in DSBs level was noticed in both cell lines. With docetaxel, an elevated DSBs level was observed in the H103 cell line. Concomitant use of hTERT gene expression depletion and administration of docetaxel effectively inhibited proliferation of FaDu cells, thus undermining successful DSBs analysis. In the case of using siRNA-based strategy in FaDu cells, irradiation (1 and 2 Gy) led to an increase in phosphorylation of the γH2AX marker compared to control on day 3. On day 7, the DSBs level was not dependent on radiation dose. A similar phenotype was observed in H103 cells.
The results obtained in the study confirm the observations made by Takahashi et al. (2014). Using therapy with telomerase-specific oncolytic adenovirus OBP-301, the authors demonstrated enhanced response to radiation in laryngeal, tongue, and pharyngeal cancer in vitro 5 . A correlation between reduced telomerase activity and efficient response to radiotherapy was also demonstrated by Ogawa et al. (1998) 34 . Observations made in this study are also supported by experiments carried out on different cancer models. Similar results were obtained by Chen et al. (2012), using siRNAs directed against the hTR subunit in cervical cancer 35 . The magnification of response to radiotherapy has also been reported in the esophageal cancer 36 and lung cancer models 37 . The results obtained in this work suggest that hTERT gene silencing results in an increased level of DSBs, which can be further magnified by simultaneous administration of ionizing radiation and/or cytostatics.
The demonstrated effect of hTERT gene silencing on DSBs production, cell cycle arrest, and apoptosis induction suggests the possibility of activation of DNA damage repair mechanisms. Up to date, literature data only describe cell cycle arrest impact on activation of individual DDR mechanisms 38 . There are still no sufficient data concerning the effect of the hTERT gene silencing using RNA interference on the activation of these mechanisms in cancer cells. This is the first time when such an analysis has been performed.
Significant increase in expression of CSB and XPA genes demonstrating activation of the NER mechanism have been reported in both FaDu and H103 cell lines. Elevated expression of the MSH6 gene (accounting for MMR mechanism) at the transcription level and an increase in XRCC4 gene expression (NHEJ mechanism) at the protein level in the FaDu cell line was also observed. Increased expression of the XRCC1 (BER) and PRKDC (NHEJ) genes on day 7 was also shown in H103 cells. The results suggest that hTERT silencing results in activation of NER as the main mechanism of DNA repair. Therefore, it can be assumed that the inhibition of activated DDR mechanisms in combination with the knockdown of the hTERT gene by RNA interference can strengthen the therapeutic effect.

Conclusions
Results reported in this study indicate that a combined approach (chemotherapy, radiotherapy, gene therapy) may be significantly beneficial in reducing chemotherapy doses, thus shortening the hospitalization period and improving patients' quality of life.

Materials and Methods
Plasmids construction. shRNA constructs were created by annealing of designed complementary oligonucleotides (sense: AATTCCCGAACACCAAGAAGTTCATCTTCAAGAGAGATGAACTTCTTGGTGTTCTTTTTG; antisense: CGCGCAAAAAGAACACCAAGAAGTTCATCTCTCTTGAAGATGAACTTCTTGGTGTTCGGG) (containing EcoRI and MluI sites) and inserting them into the EcoRI and MluI sites of pLV-THEM-GP1 vector as previously described 39 . All DNA constructs were verified using a controlled digestion with EcoRI and XbaI enzymes (both ThermoFisher Scientific, MA, USA). The shRNAs were synthesized by Sigma Aldrich (MI, USA). Lentiviral vector production, transduction and titration. Lentiviral vector plasmids encoding the designed hairpins against hTERT gene were derived by cloning into pLV-THEM-GP1 lentiviral vector, which expresses the GFP gene under control of the H1 promoter (based on plasmid #12247; Addgene; Cambridge, MA, USA). Lentiviral vectors production, transduction and titration were performed according to the protocols described previously 39,40 . The vector was produced by using of 2 nd generation system, and co-transfection of lentiviral vector plasmid (pLV-THEM-shTERT), and packaging plasmids psPAX2 (Plasmid #12260, Addgene) and pMD2.G (Plasmid #12259, Addgene) with CaCl 2 . After four days, the lentiviral vector containing supernatant was collected, filtered (0.45 μm), concentrated, and aliquots were stored at −80 °C as previously described 39 . To assess the viral copy number at an integrated DNA level, the lentiviral vector titer was measured via SYBR green-based (Roche, Germany) real-time qPCR by means of WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) method and albumin genes as templates, as described previously 40

Cell migration assay in vitro.
Cell migration was evaluated using the wound healing assay 41 . Briefly, FaDu and H103 cells were plated in a 12-well plate at a concentration of 0.2 × 10 6 cells/well, transfected and allowed to form a confluent monolayer for 72 hours. Prior to wounding, cells were starved in serum-free medium for 20 hours. Next, a 200 µl pipette tip was used to create the wound. Cell migration was observed by microscopy (plates suited in racks in exactly the same positions [coordinates]) for up to 96 h in 24-h intervals and analyzed using ImageJ (National Institutes of Health, NY, USA). The percentage of wound closure was calculated using the following formula: where: A(0) is the initial wound area, A(t) is the wound area at indicated time t.

Real-Time PCR analysis. Assessment of individual genes expression.
Quantitative analysis of gene expression was assessed using qPCR. Briefly, total RNA was extracted with TRI Reagent according to the manufacturer protocol (Sigma-Aldrich, MI, USA) 42 . cDNA was synthesized with iScript cDNA Synthesis Kit (Bio-Rad, CA, USA), using 500 ng of total RNA, oligo dT primers, and random hexamer primers. The real-time polymerase chain reaction for individual gene expression analysis was carried out using LightCycler 96 with specific primers ( Telomere length assessment. DNA was extracted from cancer cells using Wizard Genomic DNA Purification Kit (Promega, WI, USA) according to manufacturer's protocol, and stored at −20 °C. Telomere length was assessed using two pairs of primers i.e. telomere-specific and a single copy gene-specific (albumin), as previously described [43][44][45] . Briefly, we used the primers that were already shown to work specifically with conditions providing efficiency close to 100%. Initial denaturation and polymerase activation (hot start) was performed in 95 °C for 5 min. The signal was detected during 45 cycles i.e. 95 °C/10 s, 60 °C/15 s and 72 °C/10 s. Melting analysis (65-95 °C range, 0.11 °C resolution) at the end of the reaction melting analysis was performed to verify specificity of the product. The telomere length was assessed using a LightCycler 96 qPCR system (Roche, Germany) and FastStart Sybr Green Master (Roche, Germany).   Statistical analysis. Two-tailed unpaired Student's t test, ANOVA, and Chi-squared test (means from 3 separate experiments) were performed for statistical analysis using GraphPad Prism (GraphPad Software, CA, USA). P values of less than 0.05 were considered statistically significant and are indicated by the (*) symbol for p < 0.05, by (**) for p < 0.01, by (***) for p < 0.001, or by (****) for p ≤ 0.0001.
Data availability statement. All data generated or analyzed during this study are included in this published article.