Esophageal and tongue cancer cell lines shared similar expression profile with cervical cancer cells, but exhibited different E7/E6 ratios
Viral genome integration resulting in disruption and loss of viral transcripts are remarkable features of HPV-mediated oncogenesis. Therefore, we examined the HPV transcription profiles in esophageal (EC109 and EC9706), tongue (Tca83) and cervical (HeLa) cancer cell lines. Relative abundance of HPV transcripts was presented in parts per million (ppm). Overall, all these cell lines expressed E6, spliced E6 (E6*), E7, E1 and L1 transcripts (Figure 1A). However, we noted that E1 transcripts were partially expressed in both EC109 and EC9706. Other HPV transcripts (E2, E4, E5, E8 and L2) were not detected in all the cell lines. These HPV genome profiling results were consistent with previous reports 36,37.
Although relative abundance of transcripts originating from actively expressed regions of the viral genome were similar among these cell lines, differences in E6 and E7 transcript levels among cells were noted based on FPKM values. HeLa cells exhibited the highest level of HPV18 E6 transcripts (115,690), followed by Tca83 (98,246), EC9706 (71,897) and EC109 (70,874) (Table 1). Whilst spliced E6 variant I (E6*I) and E7 were markedly higher in EC109, EC9706 and HeLa (E6*I: 412,299 - 491,899; E7: 599,610 - 626,397) compared to Tca83 (E6*I: 293,362; E7: 457,654) (Table 1). Furthermore, the E7 to E6 ratios in EC109 and EC9706 were nearly doubled relative to those in HeLa and Tca83 (Figure 1B), whereas HeLa and Tca83 showed nearly doubled E6:E6*I ratios compared to EC109 and EC9706. Overall, these data revealed that while HPV18 genomes exhibited similar expression profiles in the esophageal, tongue and cervical cell lines examined, subtle differences in E6 and E7 expression patterns were noted.
HPV18 E6 in EC109, EC9706 and Tca83 targets p53 and its downstream targets in a similar manner
Following the differential expression of HPV18 oncoproteins reported above, we next examined whether E6 and E7 oncoproteins in esophageal (EC109 and EC9706) and tongue (Tca83) cancer cells target key cellular proteins in a similar manner to cervical cancer cells, such as HeLa. The cells were transfected with siRNA against HPV18 E6 and E7 (si18E6/E7). After 72 h, total protein was extracted and the levels of proteins targeted by E6, including p53, p21 and hDlg, were analyzed via Western blotting.
We found that HPV18 E6 in all tested cell lines behaved similarly in perturbing its major target, p53, but not PDZ protein. We found that, like HeLa [Fig2 A and B (i)], downregulation of HPV18 E6 in all the esophageal (EC109 and EC9706) and tongue (Tca83) SCC cell lines resulted in a significant rescue of p53 as well as its downstream transactivation target, p21 [Fig2 A and B (ii, iii and iv)]. In addition, we observed increased levels of hDlg (a PDZ protein), in HeLa cells [Fig2 A and B (i)] upon depletion of E6, but not in the esophageal and tongue SCC cell lines examined [Fig2 A and B (ii, iii and iv)].
pRB is not the major target of HPV E7 in EC109, EC9706 and Tca83
As expected, we observed that downregulation of HPV18 E6 and E7 oncoproteins led to rescue of E7 targets (pRB, p130 and p107) in HeLa cells [Fig2 A and B (ii, iii, iv)]. However, there was no significant change in the levels of pRB when E7 was downregulated in the esophageal (EC109 and EC9706) and tongue (Tca83) cell lines [Fig2 A and B (ii, iii, iv)]. We observed significantly increased levels of p130 in both EC109 [Fig2 A and B (ii)] and EC9706 [Fig2 A and B (iii)], and increased p107 was only found in EC9706 [Fig2 A and B (iii)]. Furthermore, downregulation of E7 in Tca83 did not affect the levels of pRB and its related pocket proteins [Fig2 A and B (iv)].
RB1, RB2 and p53 transcripts were not mutated in EC109, EC9706 and Tca83
As we found that downregulation of HPV18 E6 and E7 had no effect on the E7 major target protein, pRB, in esophageal (EC109 and EC9706) and tongue (Tca83) cell lines, we further analyzed our RNA-seq data to look at FPKM values of RB1 (encoding for pRB), RB2 (encoding for p130) and TP53 (encoding for p53) transcripts in HeLa, EC109, EC9706 and Tca83. As shown in Table 1, expression of RB1, RB2 and TP53 in all these cell lines were comparable for all these HPV-positive cells.
We further examined whether these transcripts harbored mutations that could potentially lead to amino acid alterations, and subsequently affect E7-pRB recognition in EC109, EC9706 and Tca83 compared to HeLa. We observed that RB2 harbored same-sense mutations, corresponding to amino acid positions at T694, R679 and T864, while no exonic mutation was detected within RB1 (Supplementary Table 1). On the other hand, we found all the cell lines carried the most common TP53 polymorphism converting Proline at amino acid codon 72 to Arginine (P72R) (Supplementary Table 1), which is consistent with previous reports 38–40.
Tca83 cells, but not EC109 and EC9706, resemble HeLa cells in targeting ERK1/2 and MMP2 signaling pathways
It is known that HPV18 oncoproteins can exert their oncogenic properties through targeting AKT 29, extracellular signal-regulated kinase (ERK) 30 and metalloprotease (MMP) 31,32 pathways in cervical cancer cells, leading to cell survival, proliferation and metastasis. To date, the involvement of HPV18 oncoproteins in perturbing these pathways in esophageal and tongue SCC cell lines has not been clearly defined. This prompted us to look at the levels of AKT, ERK 1/2, MMP2 and MMP9 activities in esophageal (EC109 and EC9706) and tongue (Tca83) cells. In general, we observed a higher basal level of both total and phosphorylated AKT at position S473 [pAKT(S473)], ERK 1/2 phosphorylated at position T202/Y204 [pERK1/2(T202/Y204)], MMP2 and MMP9 in EC109, EC9706 and Tca83 compared to HeLa cells (Fig. 3A). Despite this, we found that Tca83 cells had similar behavior to HeLa cells in targeting ERK and MMP2 pathways, while both EC109 and EC9706 cells were distinct in targeting these pathways through HPV18 oncoproteins.
When HPV18 E6 and E7 in Tca83 cells were depleted using siRNA, we observed significant reduction in pERK1/2 (T202/Y204) and MMP2, together with a significant elevation in ERK1/2 in Tca83 [Fig. 3A, B (iii-v)]. These changes were also observed in HeLa cells. While MMP9 was markedly increased in HeLa cells, no significant change was observed in Tca83 cells [Fig. 3A, B (vi)].
Meanwhile, EC109 and EC9706 cells appeared to be different from HeLa cells. Downregulation of E6 and E7 resulted in a dramatic reduced level of AKT in EC109, but not in the other cells [Fig. 3A, Fig. 3B (i) and (ii)]. In addition, E6 and E7 downregulation had no significant effect on ERK activity, MMP2 and MMP9 levels in EC109 and EC9706. These results revealed that Tca83 had similar behavior to HeLa cells in regulating ERK1/2 activity and MMP2, and both esophageal SCCs were distinct from Tca83 and HeLa cells. Nevertheless, HPV18 oncoproteins appeared to perturb AKT activity in EC109 cells.
Both Tca83 and HeLa cells require HPV18 oncoproteins to regulate the caspase pathway and proliferate
HeLa cells are addicted to HPV oncoproteins to survive 41, partly through suppression of the caspase pathway 42,43. We investigated whether this was reproducible in esophageal (EC109 and EC9706) and tongue (Tca83) cells using afore described siRNA approach to deplete E6 and E7.
We first looked at the levels of initiator (caspases 8 and 9) and effector (caspase 3) caspases. It has been shown that caspase 8 and 9 respond to extracellular apoptotic stimuli 44 and intracellular apoptosomes, respectively. This, in turn, leads to proteolytic and activation of effector caspases, including caspase 3 45. Our results showed that ablation of E6 and E7 in HeLa led to a significant increased levels of full length caspases 8, 9 and 3 [Fig. 4A, 4B (i), (iii) and (v)], as well as cleaved caspases 8 and 9 [Fig. 4A, 4B (iv) and (vi)]. Interestingly, we also observed increased levels of full length and cleaved caspases 8 and 9 in Tca83 upon depletion of HPV oncoproteins, indicating activation of caspases 8 and 9 [Fig. 4A, 4B (iii) to (vi)]. However, we did not observe activation of these initiator and effector caspases in EC109 and EC9706 (Fig. 4 and B). These results indicated that, like HeLa cells, E6 and E7 can suppress the caspase cascade in Tca83, but not in EC109 and EC9706.
We then wanted to know if esophageal and tongue SCC cells are dependent on HPV oncoproteins to proliferate. After E6 and E7 were depleted, we performed immunofluorescence assays to study the levels of Ki67 expression, a commonly used proliferation biomarker for cervical cancer. We also co-stained the cells with p53 as a measure of siRNA efficiency against HPV18 E6 and E7. Consistently, we observed a significant increased level of p53 upon depletion of HPV18 E6 and E7 in all HPV18-positive cells [Fig. 5A and B (ii)]. We observed a significant reduction in Ki67 expression in HeLa, EC9706 and Tca83, but not in EC109 upon ablation of E6 and E7 [Fig. 5A and B (i) and (ii)]. It is worth noting that Ki67 expression was relatively lower in HKESC01 than in other HPV-positive cells. Our results indicated that E6 and E7 promote proliferation of EC9706 and Tca83. Surprisingly, ablation of E6 and E7 was not adequate to initiate activation of caspase pathway in both EC109 and EC9706, as well as did not affect proliferation of EC109.
Overall, our data showed that, alike HeLa cells, Tca83 cells depend on HPV oncoproteins to attenuate initiator caspases and proliferate. In contrast, EC109 and EC9706 cells did not depend on HPV18 E6 and E7 to stimulate apoptosis. However, EC9706 cells require HPV oncoproteins to proliferate.