Although persistent infection with HPV 16/18 is regarded as the most important cofactor for CC progression [2, 3], it is insufficient on its own. Emerging evidence has indicated that the TME is a critical contributor to cancer progression [6–10]. In the TME, cancer cells can remodel their unfavorable microenvironment to support continuous and sustained proliferation, angiogenesis and metastasis [6–10]. Therefore, investigations of the detailed cellular and molecular events, especially how HPV 16/18 mediate cancer-TME communication and reshape the TME, are urgently needed. In the present study, our in vitro and in vivo experiments revealed that EVs mediated crosstalk between HPV 16/18-positive CC cells and HUVECs to remodel the TME for HPV E6-induced CC progression. By means of EVs, E6-regulated EV-Wnt7b could be transported from HPV 16/18-positive CC cells to recipient HUVECs and then modulate the HUVECs to take on a more proliferative and proangiogenic phenotype by acting on the β-Catenin signaling pathway.
EVs are increasingly recognized as critical contributors in cancer progression. They are potent mediators of communication between cancer cells and their surrounding microenvironment, which contains various types of host cells, including HUVECs [16–23, 34]. Given that E6 oncoproteins are the critical drivers of HPV 16/18-associated CC development [35, 36], we hypothesized that HPV 16/18 E6 influences the TME via EVs. As expected, we observed that EVs derived from the four HPV 16/18 E6-KD cell lines and corresponding control cells were effectively internalized by HUVECs. Moreover, compared with those of HUVECs treated with EV/E6-NC, the proliferative and proangiogenic abilities of HUVECs treated with EV/E6-KD were significantly inhibited both in vitro and in vivo. These findings suggest that EVs serve as important messengers between HPV 16/18-positive CC cells and HUVECs. Via EVs, the HPV E6 oncogene could render recipient HUVECs to take on a more proliferative and proangiogenic phenotype for CC progression.
The cargos, including proteins, DNAs, RNAs, and signaling molecules, contained in EVs are known to be transported between cells and to be responsible for EV function [19–23]. As classic signaling molecules, EV-shuttled Wnt signaling molecules, such as Wnt 11, Wnt3a, Wnt5b, and Wnt4, have been reported to be involved in cancer progression [27–30]. In the present study, we noticed that Wnt7b mRNA was highly expressed in the 4 HPV 16/18-positive CC cell lines and their EVs compared with HPV-negative cells and their EVs, respectively. EV-Wnt7b was regulated by the HPV 16/18 E6 oncogene and could be transported from HPV 16/18-positive CC cells to recipient HUVECs. Moreover, we overexpressed Wnt7b in 4 HPV 16/18 E6-KD cell lines and isolated their EVs, which restored the HUVEC proliferation and angiogenesis inhibited by E6 KD. Altogether, these results suggest that EV-shuttled Wnt7b may be responsible for HPV 16/18 E6-induced CC proliferation and angiogenesis.
Accumulating evidence has demonstrated that Wnt/β-catenin signaling is required for malignant cancer transformation [37, 38]. Once Wnt signaling is activated, β-catenin accumulates and is stabilized in the cytoplasm and translocates into the nucleus to act as a cofactor in regulating the transcription of various genes involved in cancer progression [37, 38]. Inspired by this knowledge, we therefore tested β-catenin signaling in HUVECs. We found that EV/E6-KD-treated HUVECs exhibited less nuclear β-catenin expression than corresponding controls, while treatment with EV/E6-KD + Wnt7b-OE restored the nuclear translocation of β-catenin in HUVECs. Moreover, both in vitro and in vivo results showed that treatment with FH535, an inhibitor of Wnt/β-catenin signaling [39] abolished EV-mediated HPV E6-induced HUVEC proliferation and angiogenesis. Collectively, these results suggest that EV-Wnt7b can mediate HPV E6-induced CC proliferation and angiogenesis by acting on the β-catenin signaling pathway.
Currently, emerging evidence suggests that EV-RNAs are appealing biomarkers for cancer diagnosis and prognosis because they are stable, shielded against RNase degradation and noninvasive [40, 41]. Considering the critical roles of EV-Wnt7b in CC proliferation and angiogenesis, we raised the possibility that circulating EV-Wnt7b might serve as a potential biomarker for CC prognosis. As expected, serum EV-Wnt7b levels were elevated in CC patients and significantly related to an aggressive CC phenotype. Moreover, survival analyses revealed that elevated serum EV-Wnt7b levels were related to reduced OS and RFS and that serum EV-Wnt7b is an independent prognostic factor for both OS and RFS. Notably, we successfully established a novel predictive nomogram utilizing serum EV-Wnt7b, which further confirmed that serum EV-Wnt7b had the potential to be a predictive biomarker for CC prognosis.
A major limitation of this study was that the sample size was small. Therefore, a multicenter, large-sample study should be performed to verify the predictive value of serum EV-Wnt7b in CC prognosis. In addition, our future studies should focus on exploring the exact molecular mechanism of the “HPV E6/EV-Wnt7b/β-catenin” pathway. Moreover, whether the known “HPV E6/p53” pathway interacts with the “HPV E6/EV-Wnt7b/β-catenin” pathway will be a meaningful area for further exploration.