The epidemiology of lung cancer remains partially unresolved since vast majority of smokers do not develop such tumors while at least 10–15% of lung cancers occur in non smokers 
. Thus, factors other than smoking may also have an impact as etiological and risk factors for lung cancer. Our previous studies suggested that HPV may be involved in lung cancer development through up-regulating the expressions of IL-6, IL-17 and the anti-apoptotic Mcl-1, and result in promotion of cancer cell growth 
. In addition to inactivating p53 tumor suppressors and promoting the expressions of cell proliferation proteins, HPV infection is also associated with increased tissue angiogenesis mediated by inducing the expression of various pro-angiogenic molecules including IL-8 and MMPs in cervical cancer 
. Accordingly, the goal of the present study was to determine whether HPV infection in lung adenocarcinoma cells can promote the expression of angiogenesis- and metastasis- related proteins, including IL-8 and MMPs, to make the transformed cells equipped with angiogenic and metastatic characteristics.
Our results demonstrate that HPV E6 antigens alone significantly up-regulate IL-8 expression ( & Table S1
). In addition, not only the expression of MMP-2 and MMP-9, but also their corresponding gelatinase activities are increased ( & Table S2
). Our data further reveal that the increased MMP-2 and MMP-9 expressions are mediated by the HPV E6-induced IL-8 because the MMPs levels are reduced when HPV-induced IL-8 is inhibited by combined treatment of IL-8 siRNA and neutralizing antibodies (). These observations support previous studies in which IL-8 is up-regulated following expression of HPV 16 E6/E7 in primary foreskin keratinocytes and cervical cancer cell lines 
. Besides, our results also indicate that, in addition to cell proliferation-related machinery, HPV infection can promote the expression and activities of angiogenic and metastatic molecules in lung cancer cells. The combination of growth-promoting, angiogenic and metastatic machineries further confer the malignant and tumorigenic potentials on the infected cancer cells.
Inactive p53 and high-risk HPV infection are both associated with changes in angiogenesis by a p53-dependent pathway 
. The increased angiogenesis and down-regulated apoptosis further facilitate tumor progression. On the contrary, HPV-regulated angiogenesis can alternatively occur through a p53-independent mechanism: the induction of VEGF transcription by HPV E6 remains equal in p53−/−
. The present observation that HPV 16 E6 in p53−/−
IL-6-null H1299 cells can significantly up-regulate pro-angiogenic MMPs expressions through IL-8 and our previous reports in which E6 can induce IL-17 expressions 
indicate that HPV 16 E6 can mediate angiogenic promotion and antiapoptotic effects through cytokines by a p53-independent mechanism.
IL-8 is a potent angiogenic factor and autocrine growth factor in several human cancers 
, which is associated with metastasis 
. IL-8 promotes human lung cancer growth through its angiogenic properties 
. Its autocrine and paracrine function has been shown to play an important role in angiogenesis, tumor growth, and metastasis 
. Recent reports suggest that IL-8 regulates the expression and activity of MMP-9 in human prostate carcinoma cell lines 
. Moreover, IL-8 blocking antibody is able to down-regulate MMP-9 expression and activity in orthotopic bladder cancer xenografts 
. IL-8 can also stimulate neutrophils to release stored MMP-9, which has been shown to exert positive feedback control by cleavage of IL-8, thus increasing its chemotactic activity for neutrophils 
. Expression of IL-8 by human melanoma cells up-regulates MMP-2 activity to increase tumor growth and metastasis 
. IL-8 promotes the invasiveness of endometrial stromal cells to the extracellular matrix by upregulating MMP-2 and MMP-9 activity 
. Furthermore, IL-8 is involved in the degradation of extracellular matrix by MMP-2 and MMP-9 activity, leading to endothelial cell migration, invasion and capillary tube organization 
. Our results, in support of the above findings, suggest that IL-8 may act as an autocrine and/or paracrine angiogenic factor for HPV16 E6-expressing H1299 cells by enhancing MMP-2 and MMP-9 expression.
Although abundant evidences demonstrate that IL-8 expression is induced by HPV infection in several cancer cell lines, controversial results still exist. Huang et al. reported that HPV 16 E6 down-regulates IL-8 expression through NF-k
B pathway in primary keratinocytes, which is contradictory to our observations 
. Two possible factors may explain this discrepancy: First of all, the cell model in their study is primary cell while ours is lung adenocarcinoma. The nature or difference of these 2 cell models, and thus the regulatory machineries and gene compositions, may result in the conflicting results. Secondly, the cell-type specific gene expression patterns in response to a particular pathogen may lead to the differential results. Nevertheless, HPV 16 E6 has been reported to contribute to the induction of several pro-angiogenic factors, including IL-8, in human foreskin keratinocytes 
and human umbilical vein endothelial cells 
. Particularly, our observation supports a recent report from Li et al. 
, in which HPV 16 E6 overexpression in lung cancer cells significantly promotes angiogenesis via enhancing the expression of several pro-angiogenic factors such as HIF-1a, VEGF and IL-8.Earlier studies suggest that IL-8-mediated inhibition of apoptosis could be dependent or independent of Bcl-2 expression 
. IL-8 might regulate angiogenesis by modulating the anti-apoptosis pathway of CXCR1- and CXCR2- expressing endothelial cells 
. Additionally, it is known that the anti-apoptotic Mcl-1 can also be up-regulated at both transcript and protein levels by IL-8 
, and the loss of Mcl-1 expression in human polymorphonuclear leukocytes promotes apoptosis 
. Some studies have demonstrated that IL-8 can regulate polymorphonuclear leukocytes apoptosis by up-regulating Mcl-1 antiapoptotic effect 
. These data may suggest that IL-8 might also have anti-apoptotic activity, as the scenario of IL-6, by promoting Mcl-1 expression through its autocrine and/or paracrine functions by CXCR1 and/or CXCR2 receptor in HPV16 E6- expressing H1299 cells. However, this speculation awaits further study.
In summary, our results reveal that the HPV 16 E6 significantly up-regulates pro-angiogenic MMP-2 and MMP-9 expressions through inducing IL-8 in a p53-independent manner. The present study suggests that HPV infection may be involved in angiogenic promotion mediated by IL-8, MMP-2, MMP-9 up-regulation in lung adenocarcinoma. Combining the present study and our previous data 
, a model for high-risk HPV infection to potentiate the proliferating and angiogenic activities in lung cancer cells is proposed as depicted in . First of all, HPV infection induces IL-6 and Mcl-1 expression, which subsequently promotes lung tumorigenesis. Therefore, the HPV-related lung tumorigenesis is suggested to be associated with the up-regulation of anti-apoptotic Mcl-1 expression mediating by IL-6 (black pathway in ) 
. In addition, HPV 16 E6 oncoproteins induce IL-17 levels which subsequently promote the expression of anti-apoptotic protein Mcl-1 bypassing the IL-17-downstream IL-6 molecules through PI3K pathway in a p53-independent manner. The HPV-mediated IL-17 and Mcl-1-dependent anti-apoptotic effects may play an important role in HPV-associated lung tumorigenesis (red pathway in ) 
. Thirdly, HPV-induced IL-8 may act as an autocrine and/or paracrine angiogenic factor which augments MMPs production and activities (blue pathway in ). In addition, we speculate that HPV infection may elicit a cytokine cascade by up-regulating IL-6 and IL-8 production through inducing IL-17, which is known to induce the release of IL-6 and IL-8 through the p38 and ERK MAPK pathway in human bronchial epithelial cells (green pathway in ) 
. However, this speculation needs further investigation. Taken altogether, our studies provide the evidence of an association between inflammation and HPV infection in lung cancer. The cytokines induced by HPV may work together and contribute to potentiate the proliferation and angiogenesis required for lung tumorigenesis.
Elevated cytokines induced by HPV contribute to potentiate the proliferation and angiogenesis required for lung tumorigenesis.