Roles of the PDZ-binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes

Abstract

The high-risk human papillomavirus E6 proteins have been shown to interact with and lead to degradation of PDZ-domain-containing proteins through its carboxy-terminal motif. This PDZ-binding motif plays important roles in transformation of cultured cells and carcinogenesis of E6-transgenic mice. However, its biological effects on the natural host cells have not been elucidated. We have examined its roles in an in vitro carcinogenesis model for cervical cancer, in which E6 and E7 together with activated HRAS (HRAS^G12V ) can induce tumorigenic transformation of normal human cervical keratinocytes. In this model, E6Δ151 mutant, which is defective in binding to PDZ domains, almost lost tumorigenic ability, whereas E6SAT mutant, which is defective in p53 degradation showed activity close to wild-type E6. Interestingly, we found decreased expression of PAR3 in E6-expressing cells independently of E6AP, which has not been previously recognized. Therefore, we knocked down several PDZ-domain containing proteins including PAR3 in human cervical keratinocytes expressing E7, HRAS^G12V and E6Δ151 to examine whether depletion of these proteins can restore the tumorigenic ability. Single knockdown of SCRIB, MAGI1 or PAR3 significantly but partially restored the tumorigenic ability. The combinatorial knockdown of SCRIB and MAGI1 cooperatively restored the tumorigenic ability, and additional depletion of PAR3 further enhanced the tumorigenic ability surpassing that induced by wild-type E6. These data highlight the importance of the carboxy-terminal motif of the E6 protein and downregulation of PAR3 in tumorigenic transformation of human cervical keratinocytes.

Keywords: Cervical cancer; E6; PDZ domain; human cervical keratinocytes; human papillomavirus.

Figure 1

Establishment of HCKT‐E7‐HRAS^{G 12V} expressing the wild type HPV16E6 or its mutants defective for degradation of p53 and/or PDZ domain containing proteins. Expression of HPV16E7, HRAS mutant, HRAS^{G 12V}, and HPV16E6 or its mutants was introduced to HCK1T cells by retrovirus mediated transduction as described in Materials and Method. The levels of the wild type 16E6 and its mutant were examined by Western blots (a) and RT‐PCR (b). (a) The mouse monoclonal antibody for HPV16E6 was raised against 16 amino acids of its N‐terminal region and therefore does not react with HPV16E6SAT which contains R8S/P9A/R10T substitution. While the wild type E6 and E6Δ151 which lacks its C‐terminal amino acid induced p53 degradation, E6 mutants with SAT substituions such as E6SAT and E6SATΔ151 did not do so. The levels of PDZ domain containing proteins, such as Scribble (SCRIB), DLG4, MAGI‐1 (MAGI) and PAR3 were decreased in wild‐type E6, but not E6Δ151 or E6SATΔ151 expressing cells. The level of HPV16E7 and HRAS were not affected by the expression of E6. α‐tubulin was detected as a loading control. (b) mRNA levels of the wild type E6 and its mutants were comparable in those cells. 36B4 mRNA was also detected as an internal control. (c) The level of PTPN13 was compared in indicated cells by Western blotting.

Figure 2

Tumorigenic potentials of HCK1T‐E7‐HRAS^{G 12V} expressing the wild type or E6 mutants defective for inducing degradation of p53 and/or PDZ domain containing proteins. The ability of E6 mutants to promote cell proliferation was compared to the wild type by examining growth curve (a) and clonogenic potential (b and c). HCK1T‐E7‐HRAS^{G 12V} cells transduced with retrovirus containing an empty vector (vector) or expressing the wild type or mutants of E6 were seeded at 2× 10⁴ cells/well in 12 well plates at day 0 and cell number in each well was counted at indicated time points. ** indicates P‐value < 0.05 compared to the wild type E6 expressing cells. (b, c) The cells were seeded on 35‐mm dishes under sparse conditions. After cultivating for 2 weeks, the cells were stained with Giemsa's dye, and number of colonies was counted. The photographs are representative dishes (b), and the graph illustrates means + SDs (c). Anchorage independent growth of HCK1T‐E7‐HRAS^{G 12V} expressing wild type or mutants of E6 was examined (d, e). The cells were seeded onto soft agar at 5 × 10⁴ and cultivated for 4 weeks. The representative images are shown (d). Colonies whose size was >50 μm in diameter were counted and the total number of colonies in a 16 mm² area was compared (e). Tumor promoting potentials of the E6 mutants were compared to the wild type by mouse xenografts. 1 × 10⁶ cells mixed with Matrigel were subcutaneously injected into nude mice. Mice were sacrificed at 40 days after the injection and tumor weights were compared (f). P‐value was evaluated by student's t‐test.

Figure 3

The effect of knock‐down of PDZ domain containing proteins on the tumorigenicity of HCK1T‐E7‐HRAS^{G 12V} expressing E6Δ151. Retrovirus expressing shRNA to the indicated PDZ‐ domain containing proteins was infected to HCK1T‐E7‐HRAS^{G 12V} expressing E6Δ151 at (a, c). At least two independent shRNA to each PDZ‐containing protein were tested for their efficiency to knockdown target proteins and the most efficient shRNA was used for further analysis. A retrovirus expressing shRNA to luciferase was used as a control. For knockdown of multiple target proteins, three retroviruses each expressing shRNA to either PDZ proteins or luciferases were simultaneously infected to the HCK1T‐E7‐HRAS^{G 12V} expressing E6Δ151 at MOI 5 (b, d). (a) Western blot analysis showing knockdown efficiency of shRNA to the indicated target proteins used in the following analysis. α‐tubulin was also detected as a loading control. CBB staining was performed to indicate equal loading. (b) Anchorage independent growth of HCK1T‐E7‐HRAS^{G 12V} expressing E6Δ151 with shRNA either to control or PDZ containing proteins was analyzed compared to HCK1T‐E7‐HRAS^{G 12V} expressing wild type E6 with control shRNA. (c) Tumor promoting potentials of HCK1T‐E7‐HRAS^{G 12V} expressing E6Δ151 with knockdown of single PDZ protein was compared to HCK1T‐E7‐HRAS^{G 12V} expressing the wild type E6 by mouse xenograft. The size of tumor was measured at the indicated time points. (d) The effect of knockdown multiple PDZ containing proteins on tumorigenicity of HCK1T‐E7‐HRAS^{G 12V} expressing E6Δ151 was also analyzed. Error bar indicates SD. P‐value was evaluated by student's t‐test.

Figure 4

The protein levels of PDZ containing proteins in cervical cancer cell lines. (a) The levels of PDZ containing proteins in HPV positive‐cervical cancer cell lines were compared to that in HPV negative‐cervical cancer cell lines or normal human keratinocytes by Western blot analysis. HaCaT cells are spontaneously immortalized human foreskin keratinocytes. (b) The effect of shRNA to E6AP on the level of PAR3 in cervical cancer cells were examined by Western blot analysis. HeLa, SiHa and C33a cells with E6AP shRNA was generated as described previously. p53 was included as a positive control because an E6 and E6AP complex was known to induce degradation of p53. α‐tubulin was also detected as a loading control. (c) The ability of E6 to induce reduction of PAR3 level was compared between high and low risk HPVs. The expression E6 of high risk HPVs shown in red (HPV16, 18, 26, 30, 31, 33, 35, 39, 45, 52, 56, 58 and 59 or low‐risk HPVs shown in blue (HPV6, 11, 43 and 54) was introduced to HCK1T by retrovirus mediated transduction. A retrovirus carrying empty vector was used as a control (vector). The levels of p53, MAGI‐1 and PAR3 were examined by Western blot analysis.