Complementation of human papillomavirus type 16 E6 and E7 by Jagged1-specific Notch1-phosphatidylinositol 3-kinase signaling involves pleiotropic oncogenic functions independent of CBF1;Su(H);Lag-1 activation - PubMed (original) (raw)

Complementation of human papillomavirus type 16 E6 and E7 by Jagged1-specific Notch1-phosphatidylinositol 3-kinase signaling involves pleiotropic oncogenic functions independent of CBF1;Su(H);Lag-1 activation

Karthikeyan Veeraraghavalu et al. J Virol. 2005 Jun.

Abstract

We have analyzed the induction and role of phosphatidylinositol 3-kinase (PI3K) by Notch signaling in human papillomavirus (HPV)-derived cancers. Jagged1, in contrast to Delta1, is preferentially upregulated in human cervical tumors. Jagged1 and not Delta1 expression sustained in vivo tumors by HPV16 oncogenes in HaCaT cells. Further, Jagged1 expression correlates with the rapid induction of PI3K-mediated epithelial-mesenchymal transition in both HaCaT cells and a human cervical tumor-derived cell line, suggestive of Delta1;Serrate/Jagged;Lag2 ligand-specific roles. Microarray analysis and dominant-negatives reveal that Notch-PI3K oncogenic functions can be independent of CBF1;Su(H);Lag-1 activation and instead relies on Deltex1, an alternative Notch effector.

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Figures

FIG. 1.

FIG. 1.

Representative photomicrographs show expression of DSL ligands in CIN III and invasive squamous cell carcinoma of cervix (SCC) as determined by mRNA in situ hybridization (A to D) and immunohistochemistry (E to H); 11 out of 13 CIN III (A) and 14 out of 16 SCC (C) cases failed to show detectable Delta1 transcripts. (B) Detection of Delta1 transcripts in one of the CIN III cases. (D) Abundant expression of Jagged1 transcripts in SCC as reported earlier (41). Fluorescein isothiocyanate (FITC)-labeled mRNA probes and an alkaline phosphatase-conjugated anti-FITC antibody-based detection system were employed. Antisense staining is in purple (indicated by arrows) and the sections were counter stained with fast green. The FITC-labeled antisense and sense riboprobes were in vitro transcribed using SP6 or T7 primers from the cDNA templates encoding full-length Delta1 (pcDNA3-Delta1) and Jagged1 (pcDNA3-Jag1) (41). (E and G) Representative photomicrographs that show mild immunohistochemical staining of Delta1 (1:100; sc-9102; Santa Cruz Biotech) in CIN III and SCC cases, respectively. As a control for immunohistochemical staining, CIN III and SCC sections were stained for a proliferative marker protein, PCNA (Santa Cruz Biotech) (F and H, respectively). Arrows indicate areas of positive diaminobenzidine (DAB) staining. The counterstain is hematoxylin. Photomicrographs were taken under 40× magnification.

FIG. 2.

FIG. 2.

HaCaT cells stably expressing Jagged1 and hemagglutinin (HA)-tagged Delta1 were stained using anti-Jagged1 (2.5 μg/ml, sc-6011; Santa Cruz Biotech) (B) and anti-Delta1 antibody (2.5 μg/ml, sc-9102; Santa Cruz Biotech) (D). Endogenous expression of Notch1 in HaCaT cells was detected using anti-Notch1 antibody (1:50, sc-6014; Santa Cruz Biotech) (F). A, C, and E are isotype control immunocytochemical staining. Photomicrographs were taken under 40× magnification. (G) HaCaT cells stably expressing mock vector (Neo), activated Notch1 (AcN1), Jagged1 (Jag1), or Delta1 were transiently transfected with the plasmid combinations (total of 10 μg) encoding either bicistronic HPV-16 E6 and E7 (E6/E7) {pMSIIref-E6/E7} (gift of M. Conrad-Stoppler and H. Stoppler), human Manic Fringe (MFng) (pcDNA3-MFng) or neomycin (Neo) (pcDNA3.0-Neo), and injected subcutaneously into nude mice. After 3 weeks, the mice were sacrificed, and tumor sizes were measured as described previously (8). The graph shows tumor volume after 3 weeks, and each dot represents a tumor from one mouse.

FIG. 3.

FIG. 3.

(A) Representative photomicrographs (4× magnification) show the extent to which HaCaT-Neo, -Jagged1 (Jag1), and -Delta1 (Del1) cells exhibited motility in the presence of low serum (2% fetal calf serum) and covered the wounded nude area after 24 h in an in vitro wound-healing assay (26). The photomicrographs in the panel below show the extent to which CaSki and SiHa exhibit motility in a similar assay.(B) Immunoblots reveal the analysis of CaSki and SiHa cell lysates for the expression of Notch1 (1:10; bTAN20; DSHB) (WB: Notch1), Jagged1 (1:10; TS1.15SH; DSHB) (WB: Jagged1), and Delta1 (1:300; sc-9102; Santa Cruz Biotech) (WB: Delta1). As a gel-loading control, the same blots were reprobed with β-actin antibody. M, molecular size markers. (C) Graphs represent the percent wounded nude area covered by the mentioned cell lines under different conditions. The % wound area covered was measured using a Nikon inverted microscope, and the associated Image-pro Plus software. Graph on the top and bottom show that HaCaT-Jagged1 and CaSki cells cover 90% of the wounded nude area by 24 h, respectively. Treatment with neutralizing anti-Notch1 antibody (α-Notch1 ab) (1:300; Neomarkers) or the inhibitor of presenilin-dependent γ-secretase (20 μM, GI; Calbiochem) or LY294002 (20 μM, LY; Calbiochem) or stable expression of plasmids encoding different cDNAs for soluble human Jagged1 (Sol hJag1 and pcDNA-sol Jag1) or dominant negative Akt (DN-Akt) resulted in significant inhibition of wound closure in both HaCaT-Jagged1 and CaSki cells (P < 0.001). Treatment with dimethyl sulfoxide (vehicle control) or mouse isotype immunoglobulin G or stable expression of mock vector were included as controls. The results shown represent the means ± standard deviation of the results from three independent experiments.

FIG. 4.

FIG. 4.

Representative photomicrographs (40× magnification) show alterations in the expression of EMT markers in HaCaT-Jagged1 versus HaCaT-Neo cells (A) and CaSki cells (B). Immunofluorescence staining of E-cadherin (1:300; Calbiochem), plakoglobin (1:300; Sigma), and fibronectin (1:300; Sigma) is shown in HaCaT-Neo (A, i to iii) and HaCaT-Jagged1 (A, iv to vi) cells. (B) Immunofluorescence staining of E-cadherin (B, i) and plakoglobin (B, iv) in confluent monlolayer culture of CaSki cells are shown. Downregulation of E-cadherin (B, iii) and plakoglobin (B, vi), and upregulation of vimentin (B, viii) in CaSki cells at the wound healing edge are shown. B, ii, v, and vii represent bright-field images of the wound healing edge.

FIG. 5.

FIG. 5.

(A) Graphs represent the percent wounded nude area covered in the presence of low serum (2% FCS) by the mentioned cell lines stably transfected with plasmids encoding different cDNAs (either mock vector or dominant negative CSL (DN-CSL and pcDNA3-DN CSL) or dominant negative Deltex1 (DN-Dtx1 and pEF-BOS-HA-hDxI-II). Graphs on the left and right show that mock vector-transfected (pcDNA3.0-Neo) HaCaT-Jagged1 and CaSki cells cover 90% of the wounded nude area by 24 h. Stable expression of DN-Dtx1 but not DN-CSL resulted in significant inhibition of wound closure in both HaCaT-Jagged1 and CaSki cells (P < 0.001). (B) Immunoblot on the top panel shows levels of phospho-PKB/Akt Ser473 (pAkt473) (1:1,000; Cell Signaling Technology) in HaCaT cells stably expressing mock vector or DN-CSL or DN-Dtx1. The same blot was reprobed to detect total Akt and is illustrated in the bottom panel. (C) Immunoblots show levels of Hes1 (1:300; sc-13844; Santa Cruz Biotech) and cyclin D1 (1:300; Santa Cruz Biotech). β-Actin levels show comparable protein loading across lanes. (D) Graph shows the number of colonies on soft agar generated by cell lines transfected with plasmids encoding L83V E6/E7 along with either mock vector or DN-Dtx1 or treated with GI (20 μM; Calbiochem). The data were generated by microscopic counting of colonies in 20 random fields under 10× magnification as described (33). The results shown in graphs A and D represent the means ± standard deviation from three independent experiments.

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References

    1. Amsen, D., N. J. M. Blander, G. R. Lee, K. Tanigaki, T. Honjo, and R. A. Flavell. 2004. Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells. Cell 117:515-526. - PubMed
    1. Artavanis-Tsakonas, S., M. D. Rand, and R. J. Lake. 1999. Notch signalling: cell fate control and signal integration in development. Science 284:770-776. - PubMed
    1. Ascano, J. M., L. J. Beverly, and A. J. Capobianco. 2003. The C-terminal PDZ ligand of JAGGED1 is essential for cellular transformation. J. Biol. Chem. 278:8771-8779. - PubMed
    1. Aster, J. C., L. Xu, F. G. Karnell, V. Patriub, J. C. Pui, and W. S. Pear. 2000. Essential roles for ankyrin repeat and transctivation domains in induction of T-cell leukemia by notch1. Mol. Cell. Biol. 20:7505-7515. - PMC - PubMed
    1. Bakin, A. V., A. K. Tomlinson, N. A. Bhowmick, H. L. Moses, and C. L. Arteaga. 2000. Phosphatidylinositol 3-kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition and cell migration. J. Biol. Chem. 275:36803-36810. - PubMed

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