HPV16-E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by down regulating the expression of KIF7

Background: The E6 and E7 proteins in HPV16 are the main oncogenes in the occurrence of lung cancer. In recent studies, we had found that E6 and E7 downregulated the expression of LKB1 in lung cancer cells. However, it is not clear how E6 and E7 regulate LKB1 in lung cancer cells. Methods: The double directional genetic manipulation and Nuclear plasma separation technology were performed to explore the molecular mechanism of E6 and E7 inhibiting the antitumor activity of LKB1 in well-established lung cancer cell lines. Results: E6 but not E7 significantly downregulated the expression of tumor suppressor KIF7 at protein level, and the inhibition of KIF7 further reduced the expression of LKB1 both in the nuclei and in the cytoplasm, whereas reduced the expression of p-LKB1 in the cytoplasm only. Therefore, we suggested that HPV16 E6 but not E7 downregulates the antitumor activity of LKB1 by down regulating the expression of p-LKB1 in the cytoplasm only. Conclusion: We demonstrated for the first time that E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by down regulating the expression of KIF7. Our findings provided new evidence to support the important role of KIF7 in the pathogenesis of lung cancer and suggested new therapeutic targets. of KIF7 not only significantly downregulated the expression of LKB1, but also downregulated the expression of p-LKB1. Our results from nuclear-cytoplasmic protein separation western blot analysis showed that the inhibition of KIF7 significantly decreased the expression level of p-LKB1 at Ser 428 in the cytoplasm, but the expression level in the nucleus was basically unchanged. These results indicated that the antitumor activity of LKB1 was determined by two factors, one was phosphorylation of LKB1, the other was that p-LKB1 must be located in the cytoplasm, both were indispensable. It is not clear which of the two conditions occurs first, the phosphorylation or the subcellular translocation. The detailed molecular mechanism needs further study in the future.


Background
In 1980, Syrjänen first proposed the hypothesis of the role of HPV infection in the occurrence of bronchosquamous cell carcinoma [1]. With the rapid development of molecular biology technology and its timely application in the study of lung cancer, researchers found that E6 and E7 proteins in HPV16 are the main oncogenes, and long term persistent infection is often related to the occurrence of lung cancer, especially in non-smokers and Asians [2][3][4][5]. The E6 protein inhibited cell apoptosis mainly by degrading p53 gene [6], while the E7 protein promoted cell proliferation mainly by inhibiting retinoblastoma protein (pRb) [7]. Therefore, the signaling pathways were regulated by E6 and E7 proteins might not be exactly the same in the occurrence of lung cancer. Recently, we found that the overexpression of both E6 and E7 in HPV 16 downregulated the expression of LKB1 at both protein and mRNA levels in lung cancer cells [8,9]. However, the potential molecular mechanism of regulation of LKB1 by E6 and E7 is not clear.
Liver kinase B1 (LKB1), also known as serine / threonine kinase 11 (STK11), was first found the tumor suppressor gene in patients with Peutz Jeghers syndrome (PJs) [10,11]. It has serine / threonine protein kinase activity and plays an important role in the occurrence and development of lung cancer by phosphorylating substrate protein or binding with target protein to regulate gene expression [12,13]. It had been shown that LKB1 is the key barrier of lung tumorigenesis, which controlled the occurrence, differentiation and metastasis of lung tumorigenesis [14]. The vitro experiment results showed that the phosphorylation of LKB1 at Ser 428 was involved in the anticancer effect of human oral cancer cells [15]. Recently, Wong et al. found that KIF7 promoted the antitumor activity of LKB1 through up regulation the expression of LKB1 and induction the phosphorylation of LKB1 at Ser 428 [16].
Kinesin family member 7 (KIF7) is on 15q26.1 and a Kinesin-4 family member that has been shown to play critical roles in primary cilia formation and Hedgehog (Hh) signaling in embryonic development [17,18]. KIF7 was a novel tumor suppressor in prostate cancer that acts by suppressing proliferation, migration, invasion and tumorigenesis through LKB1/PTEN/AKT signaling pathway [16], the low expression of KIF7 indicated poor prognosis in epithelial ovarian cancer [19] and facilitated cell survival and migration of choriocarcinoma cells [20].
In this study, we investigated that HPV16 E6 up-regulated the expression of p-LKB1 through HPV-KIF7-LKB1 axis. Here, we report that HPV16-E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by down regulating the expression of KIF7

Materials And Methods
Cell culture H1299 and A549 cell lines were obtained from the ATCC (Manassas, VA, USA) and cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) at 37˚C in a 5% CO 2 humidified atmosphere.

1) Transfection and interference
Based on the previous study results, H1299 cells were E6 and E7 low-expression cell lines, while A549 cells were E6 and E7 high-expression cell lines [9]. The plasmid of pEGFP-N1-HPV16 E6, pEGFP-N1-HPV16 E7, and pEGFP-N1 were kindly provided by Prof Xudong Tang, Institute of Biochemistry and Molecular Biology, Guangdong Medical College, China. HPV16 E6 SiRNA and HPV16 E7 SiRNA were purchased from RIBOBIO (Guangzhou, China), KIF7 siRNA was purchased from General Biosystems (Anhui, China). Disordered siRNA was used as a nonspecific siRNA control.
The cells were performed in 6-well plates using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) as per instructions from the manufacturer. Transfection with empty vector and mock transfection were used as controls. The protein analysis was assessed 48 hours after transfection by western blotting. The mRNA analysis was assessed 24 hours after transfection by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR).

Western-blot assays
The assays were performed as previously described [9]. Information about primary antibodies is as follows:

Quantitative real-time PCR
Total RNA was extracted from cells with Trizol solution (TaKaRa, Dalian, China) and RNase RNA isolation kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. A total of 1μg RNA was subjected to reverse transcription reaction to obtain cDNAs by using a Prime Script TM RT reagent Kit (TaKaRa, Dalian, China).
qRT-PCR was performed using SYBR ® Premix Ex Taq II (TaKaRa, Dalian, China) on 7900HT Fast Real-Time PCR System (Applied Biosystems). Non-template controls were carried out every time for each primer pair to detect nonspecific amplification. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as the internal control. All reactions were run in triplicate. The fold change of mRNA expression was calculated using the 2 −ΔΔct method. The detailed information of the primers is given in Table 1.
Nuclear plasma separation technology H1299 cells were interference with SiKIF7. Nuclear and Cytoplasmic Protein Extraction Kit (P0028, Beyotime, Shanghai, China) was used to perform nuclei isolation. All the experiments were performed on ice, Cytoplasmic protein extraction reagent A with 1mM PMSF was added to the cell suspension, and then 10-15 minutes of ice bath after 5 seconds of violent oscillation at the highest speed, cytoplasmic protein was added to cytoplasmic protein extraction reagent B. After 5 seconds of violent oscillation at the highest speed, the supernatant was centrifuged at 4℃ at 12000g for 5 minutes. The nuclear protein extraction reagent with 1mM PMSF was added to the remaining precipitate, which was intensely vortex at the highest speed for 15-30 seconds and then placed in an ice bath for 30 minutes, during which time the nuclear protein was intensely vortex for 15-30 seconds every 1-2 minutes. After centrifugation at 4℃ and 12000g for 10 minutes, the supernatant was absorbed into the precooled plastic tube, which was nuclear protein.
Statistical analysis SPSS 22.0 software was utilized for statistical analyses in this study. Each assay was performed at least 3 times. The data were expressed as mean ± SD. Statistical significance was determined by Student's t-test, and a p value<0.05 was considered significant.

Results
The screening of lung cancer cell lines Based on our previous results, H1299 cell line was low E6 and E7 expression cell line, and A549 cell line was high E6 and E7 expression cell line [21]. The expression of KIF7 was screened in normal bronchial epithelial cell line HBE and lung cancer lines H1299, H460, and A549, respectively. As shown in S1Fig, the high expression level of KIF7 was found in HBE and H1299, whereas low expression level of KIF7 was observed in H460 and A549 cell lines. Further assays were designed and performed based on these results.
The overexpression of E6 significantly downregulated the expression of KIF7 and LKB1, whereas the overexpression of E7 significantly downregulated the expression of LKB1 only.
The pEGFP-N1-E6 or E7 vectors were transiently transfected into the low expression H1299 cell line, and the E6 or E7 empty vectors and mock transfections served as controls. The results showed that the overexpression of E6 significantly downregulated the expression of KIF7 at protein level only and the expression of LKB1 at both protein and mRNA levels, whereas the overexpression of E7 significantly downregulated the the expression of LKB1 at both protein and mRNA levels and had no regulatory effect on KIF7. Results are presented in Figure1A. The inhibition of E6 evidently upregulated the expression of KIF7 and LKB1, whereas the inhibition of E7 evidently upregulated the expression of LKB1 only.
To further verify the regulatory roles of E6 or E7 on KIF7 and LKB1, we applied E6 or E7-specific siRNA to knockdown the expression of E6 or E7 in the A549 cell line. E6 or E7-nonspecific siRNA and mock specific siRNA were used to serve as the controls. The results indicated that the inhibition of E6 evidently upregulated the expression of KIF7 at protein level only and the expression of LKB1 at both protein and mRNA levels, whereas the inhibition of E7 evidently upregulated the expression of LKB1 at both protein and mRNA levels and had no regulatory effect on KIF7. The results were presented in Fig.1 B.

The inhibition of KIF7 obviously downregulated the expression levels of LKB1 and p-LKB1
To further verify the regulatory roles of KIF7 on LKB1 and p-LKB1, we applied KIF7-specific siRNA to knockdown the expression of KIF7 in the H1299 cell line. KIF7-nonspecific siRNA and mock specific siRNA were used to serve as the controls. The results indicated that the inhibition of KIF7 obviously downregulated the expression of LKB1 at both protein and mRNA levels and the expression of p-LKB1 at protein level. The results were presented in Fig.2 A. The loss of KIF7 reduced the expression of LKB1 both in the nuclei and in the cytoplasm, whereas reduced the expression of p-LKB1 in cytoplasm only.
To determine whether the localization of LKB1 or p-LKB1 is related to the regulation of KIF7, we used western blotting to detect the expression of LKB1 or p-LKB1 in nucleic and cytoplasmic fractions. We knocked KIF7 in H1299 cells and separated the proteins in the nucleus and in the cytoplasm by using nuclear plasma separation technology. We found that the expression level of LKB1 was downregulated in both the nuclei and the cytoplasm, whereas the expression level of p-LKB1 was downregulated in cytoplasm only. The results were presented in Fig.2B.

Discussion
In our previous work, we found that the overexpression of both E6 and E7 in HPV 16 downregulated the expression of LKB1 at both protein and mRNA levels in lung cancer cells [8,9]. However, the underlying molecular mechanism of the regulation of LKB1 by HPV16 is not clear. In this study, we found that the overexpression of E6 significantly downregulated the expression of KIF7 at protein level only and the expression of LKB1 at both protein and mRNA levels. Conversely, the knockdown of E6 evidently upregulated the expression of KIF7 at protein levels only and the expression of LKB1 at both protein and mRNA levels. We believed that the regulation mechanism of E6 on KIF7 might be through translational or post-translational pathways. However, we found that whether by transfection or interference, E7 had no regulatory effect on KIF7. Although E7 could also downregulate the expression of LKB1, it was not achieved by downregulating KIF7. Therefore, we speculate that the regulation of LKB1 by E7 may be accomplished by other genes. To our knowledge this is the first time found that KIF7 was involved in HPV16 E6 but not E7 downregulate the expression of LKB1 in lung cancer cells. The detailed regulatory mechanism between E6 and KIF7 needs to be further studied in the future.
The increased expression of protein and mRNA of LKB1 does not mean that its antitumor activity is increased. Wong et al. had speculated that KIF7 upregulated the antitumor activity of LKB1 in two ways, one was phosphorylation, and the other was subcellular localization; and they also found that KIF7 significantly upregulated the expression of LKB1 in both the nuclei and the cytoplasm and promoted the phosphorylation of LKB1 at Ser 428 [16]. However, they did not find that the expression level of LKB1 phosphorylation induced by Kif7 in the nucleus was completely different from that in the cytoplasm. In the present study, we found that inhibition of KIF7 not only significantly downregulated the expression of LKB1, but also downregulated the expression of p-LKB1. Our results from nuclear-cytoplasmic protein separation western blot analysis showed that the inhibition of KIF7 significantly decreased the expression level of p-LKB1 at Ser 428 in the cytoplasm, but the expression level in the nucleus was basically unchanged. These results indicated that the antitumor activity of LKB1 was determined by two factors, one was phosphorylation of LKB1, the other was that p-LKB1 must be located in the cytoplasm, both were indispensable. It is not clear which of the two conditions occurs first, the phosphorylation or the subcellular translocation. The detailed molecular mechanism needs further study in the future.
We had carried out a series of studies on the role of HPV16 in the carcinogenesis of lung cancer in the past, and the carcinogenic effects of E6 and E7 proteins were completely consistent [8,9,21]. However, Liu et al. had found that E6 and E7 in HPV16 played different roles in the regulation of ERK signaling pathway [22]. For the first time in this study, we found that HPV16-E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by down regulating the expression of KIF7.

Conclusion
We demonstrated for the first time that E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by downregulating the expression of KIF7 and the antitumor activity of LKB1 was determined by two factors, one was phosphorylation of LKB1, the other was that p-LKB1 must be located in the cytoplasm, both were indispensable. Our findings provided new evidence to support the important role of KIF7 in the pathogenesis of lung cancer and suggested new therapeutic targets.

Figure 2
The effects of KIF7 on the regulation of LKB1 and p-LKB1 expression by interference, KIF7, LKB1, and p-LKB1 were demonstrated by western blotting in H1299 cells (A). The inhibition of KIF7 downregulated the expression levels of LKB1 in both the cytoplasmic and the nuclear and the expression level of p-LKB1 in the cytoplasmic only. KIF7-specific siRNA was performed by western blotting in H1299 cells (B). Mock: mock transfection; NS: no significance (*p<0.05; **p<0.0l).

Supplementary Files
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