The PDZ binding motif of human papillomavirus type 16 E6 induces PTPN13 loss, which allows anchorage-independent growth and synergizes with ras for invasive growth - PubMed (original) (raw)

The PDZ binding motif of human papillomavirus type 16 E6 induces PTPN13 loss, which allows anchorage-independent growth and synergizes with ras for invasive growth

William C Spanos et al. J Virol. 2008 Mar.

Abstract

The human papillomavirus (HPV) oncogene E6 has been shown to perform multiple functions (p53 degradation, telomerase activation, etc.) that play a role in oncogenic transformation. Beyond known E6 functions, an undefined mechanism that allows cellular invasion requires the E6 PDZ binding motif (PDZBM). Here, we show that HPV type 16 (HPV16) E6 interacts with and induces loss of a protein tyrosine phosphatase (PTPN13) in a PDZBM-dependent manner. PTPN13 loss induced either by the presence of E6 or by a short hairpin RNA strategy allows for anchorage-independent growth (AIG) and synergy with a known oncogene, Ras(v12), resulting in invasive growth in vivo. Restoring PTPN13 expression reverses AIG in cells lacking PTPN13. A genomic analysis of colorectal carcinoma has identified an association between PTPN13 loss-of-function mutations and aberrant Ras signaling. Our findings support this correlation and provide methods for further evaluation of the mechanisms by which PTPN13 loss/Ras expression leads to invasive growth, the results of which will be important for treatment of HPV-related and non-HPV cancer.

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Figures

FIG. 1.

FIG. 1.

E6 induction of AIG and invasive growth is dependent on the PDZBM. (A) E6 induction of AIG. Single-cell suspensions of 104 primary (1°) MTECs and MTECs transduced with LXSN (vector control), E6, E6Δ (no PDZBM), H-Ras, E6/H-Ras, E6Δ/H-Ras, and HNSCC HPV− (UMSCC84) and HPV+ (UPCI SCC90) cell lines were seeded in triplicate into soft agar and photographed at a magnification of ×40 after 3 weeks. Colonies and corresponding quantifications of colony-forming efficiency (in percentages of AIG) along with standard errors (in parentheses) are shown; n = 3, with experiments repeated. (B) Average tumor growth curves for mice injected with the indicated cells subcutaneously in the flank. ▪, E6/Ras; ♦, E6; ▴, E6Δ/Ras; •, E6Δ; X, Ras. Error bars represent s.d.; n = 6. (C) Ras expression in MTECs after retroviral transduction with either vector expressing Ras. The upper panel shows total Western blotting results, comparing total Ras levels in the indicated cell lines. GAPDH levels are shown to compare protein loading results.

FIG. 2.

FIG. 2.

HPV16 E6 PDZBM is required for induction of loss and for physical association with PTPN13. The results of Western blot analyses of indicated proteins after retroviral transduction with the indicated vectors (LXSN, E6, E6Δ) are shown as follows. (A) HTECs with extended life in culture due to expression of hTert and shRNA in response to the presence of p16. (B) MTECs. (C) HNSCC lines with HPV16 (UPCI SCC90 [HPV+]) and without HPV16 (UMSCC84 [HPV−]), with E6 mRNA expression levels indicated as percents (with s.d. in parentheses); n = 3, with experiments repeated. (The E6 level shown for all cell lines refers to quantitative real-time RT-PCR of E6 mRNA standardized to an 18S rRNA signal.) (D and E) GST-E6 proteins (upper panel) bound directly to GST beads were incubated with lysates of cells of the 293 cell line (middle panel) expressing mouse or human PTPN13. Western blotting (IB) of mouse and human PTPN13 revealed coprecipitation (IP) of GST-E6 products with mouse and human PTPN13, respectively (lower panel). (F) PTPN13 quantitative real-time RT-PCR standardized to an 18S rRNA signal. Error bars represent s.d.; n = 3, with experiments repeated.

FIG. 3.

FIG. 3.

Modulation of PTPN13 alters AIG. (A) Levels of AIG with loss of PTPN13 protein in MTECs expressing E6Δ in isolated clonal populations of cells after transduction with shRNA constructs targeting PTPN13 compared to the results obtained with control (V) nontargeting shRNA vector. In all panels, standard errors are shown in parentheses; n = 3, with experiments repeated. (B) Levels of AIG with loss of shPTPN13 in early-passage MTECs cells transduced with shRNA targeting PTPN13 (clonal population) compared to the results obtained with nonsense shRNA vector (V). (C) PTPN13 in an HPV− HNSCC cancer line (UMSCC84). C, no treatment; V, nonsense shRNA vector; shRNA, vector targeting PTPN13. (D) Loss of PTPN13 in HaCaT cells, representing a skin keratinocyte, induced by the presence of either E6 or shPTPN13 increased AIG. Lysates of cells transduced with the indicated constructs were examined for AIG and loss of PTPN13. (E and F) UPCI SCC90 (HPV+) and HEK293 cell lines were transfected and selected with G418 to obtain stable lines expressing either GFP or PTPN13. AIG assay results and PTPN13 protein levels after transfection are shown. HTEC PTPN13 protein levels are shown in panel E for comparison to the amount of PNPN13 in transfected cells.

FIG. 4.

FIG. 4.

Loss of PTPN13 synergizes with H-Ras for invasive growth in immune-competent mice. (A) Average growth curves for mice injected with the indicated cells subcutaneously in the flank. ♦, E6/Ras; •, shPTPN13/Ras; ⋄, E6Δ/shPTPN13/Ras; ▪, shPTPN13; ▴, E6Δ/shPTPN13. Error bars represent s.d.; n = 6. (B) Immunohistochemistry of PTPN13 protein in normal mouse tonsil epithelium (panels a and d), E6/Ras tumors (panels b and e), and shPTPN13/Ras tumors (panels c and f) in vivo. Panels a to c and panels d to f present the results of the use of antibodies against epitopes for the C and N termini of the protein, respectively. Insets show the results obtained with either blocking peptide (panels a to c) or secondary controls (panels d to f) alone. Bars, 130 um.

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