Limits...
The invasive capacity of HPV transformed cells requires the hDlg-dependent enhancement of SGEF/RhoG activity.

Krishna Subbaiah V, Massimi P, Boon SS, Myers MP, Sharek L, Garcia-Mata R, Banks L - PLoS Pathog. (2012)

Bottom Line: We also show that HPV-18 E6 can interact indirectly with SGEF in a manner that is dependent upon the presence of hDlg and PDZ binding capacity.In HPV transformed cells, E6 maintains a high level of RhoG activity, and this is dependent upon the presence of hDlg and SGEF, which are found in complex with E6.Furthermore, we show that E6, hDlg and SGEF each directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.

ABSTRACT
A major target of the HPV E6 oncoprotein is the human Discs Large (hDlg) tumour suppressor, although how this interaction contributes to HPV-induced malignancy is still unclear. Using a proteomic approach we show that a strong interacting partner of hDlg is the RhoG-specific guanine nucleotide exchange factor SGEF. The interaction between hDlg1 and SGEF involves both PDZ and SH3 domain recognition, and directly contributes to the regulation of SGEF's cellular localization and activity. Consistent with this, hDlg is a strong enhancer of RhoG activity, which occurs in an SGEF-dependent manner. We also show that HPV-18 E6 can interact indirectly with SGEF in a manner that is dependent upon the presence of hDlg and PDZ binding capacity. In HPV transformed cells, E6 maintains a high level of RhoG activity, and this is dependent upon the presence of hDlg and SGEF, which are found in complex with E6. Furthermore, we show that E6, hDlg and SGEF each directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells. These studies demonstrate that hDlg has a distinct oncogenic function in the context of HPV induced malignancy, one of the outcomes of which is increased RhoG activity and increased invasive capacity.

Show MeSH

Related in: MedlinePlus

The hDlg/SGEF module is required for the invasive capacity of HeLa and CaSki cells.The cells (HeLa in Panel A and CaSki in Panel B) were transfected with siRNAs to Luciferase (Luc), E6/E7, hDlg or SGEF and after 72 hrs the cells were harvested and equal numbers plated onto Matrigel invasion chambers. After overnight incubation the numbers of invading cells in the lower chamber were counted. The graphs show the fold change in the numbers of invading cells from multiple assays, where siLuc- transfected cells were scored as the reference point. Error bars represent ±SD of multiple experiments. The right hand panels show the western blot analysis of the levels of expression in total cell extracts of hDlg, p53 and SGEF following siRNA transfections performed in parallel with the invasion assays. α-Actinin is shown as the loading control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3285591&req=5

ppat-1002543-g009: The hDlg/SGEF module is required for the invasive capacity of HeLa and CaSki cells.The cells (HeLa in Panel A and CaSki in Panel B) were transfected with siRNAs to Luciferase (Luc), E6/E7, hDlg or SGEF and after 72 hrs the cells were harvested and equal numbers plated onto Matrigel invasion chambers. After overnight incubation the numbers of invading cells in the lower chamber were counted. The graphs show the fold change in the numbers of invading cells from multiple assays, where siLuc- transfected cells were scored as the reference point. Error bars represent ±SD of multiple experiments. The right hand panels show the western blot analysis of the levels of expression in total cell extracts of hDlg, p53 and SGEF following siRNA transfections performed in parallel with the invasion assays. α-Actinin is shown as the loading control.

Mentions: HeLa cells are highly tumourigenic and exhibit invasive capacity in matrigel assays [49]. Based upon the above results, we reasoned that this invasive capacity might be dependent upon the active Dlg/SGEF pool that is maintained by E6/E7 expression. To investigate this we proceeded to perform a series of matrigel invasion assays using HeLa cells in which either E6/E7, hDlg or SGEF expression was ablated by siRNA transfection. Cells were transfected with the relevant siRNAs and after 72 hrs the cells were trypsinised and counted, and equal numbers of cells were inoculated into matrigel invasion chambers. At the same time, the efficiency of the siRNAs was determined by western blotting for the relevant target protein (Figure 9A). Chambers were left overnight at 37°C and the number of cells invading the lower chamber was counted the following day. The collated results from multiple assays are also shown in Figure 9A. Not surprisingly, loss of E6/E7 expression results in a dramatic decrease in the invasive capacity of these cells. However, loss of either hDlg or SGEF also results in a significant decrease in the capacity of these cells to invade the matrigel. We also repeated this assay in HPV-16 positive CaSki cells and the results shown in Figure 9B also confirm that continued expression of hDlg and SGEF contribute directly to the invasive potential of these cells. These results demonstrate that, in the context of HPV-induced malignancy, the residual levels of hDlg found within HPV-18 positive HeLa cells and HPV-16 positive CaSki cells actually contribute directly to the invasive potential of these cells, most likely through up-regulation of SGEF and RhoG activity.


The invasive capacity of HPV transformed cells requires the hDlg-dependent enhancement of SGEF/RhoG activity.

Krishna Subbaiah V, Massimi P, Boon SS, Myers MP, Sharek L, Garcia-Mata R, Banks L - PLoS Pathog. (2012)

The hDlg/SGEF module is required for the invasive capacity of HeLa and CaSki cells.The cells (HeLa in Panel A and CaSki in Panel B) were transfected with siRNAs to Luciferase (Luc), E6/E7, hDlg or SGEF and after 72 hrs the cells were harvested and equal numbers plated onto Matrigel invasion chambers. After overnight incubation the numbers of invading cells in the lower chamber were counted. The graphs show the fold change in the numbers of invading cells from multiple assays, where siLuc- transfected cells were scored as the reference point. Error bars represent ±SD of multiple experiments. The right hand panels show the western blot analysis of the levels of expression in total cell extracts of hDlg, p53 and SGEF following siRNA transfections performed in parallel with the invasion assays. α-Actinin is shown as the loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285591&req=5

ppat-1002543-g009: The hDlg/SGEF module is required for the invasive capacity of HeLa and CaSki cells.The cells (HeLa in Panel A and CaSki in Panel B) were transfected with siRNAs to Luciferase (Luc), E6/E7, hDlg or SGEF and after 72 hrs the cells were harvested and equal numbers plated onto Matrigel invasion chambers. After overnight incubation the numbers of invading cells in the lower chamber were counted. The graphs show the fold change in the numbers of invading cells from multiple assays, where siLuc- transfected cells were scored as the reference point. Error bars represent ±SD of multiple experiments. The right hand panels show the western blot analysis of the levels of expression in total cell extracts of hDlg, p53 and SGEF following siRNA transfections performed in parallel with the invasion assays. α-Actinin is shown as the loading control.
Mentions: HeLa cells are highly tumourigenic and exhibit invasive capacity in matrigel assays [49]. Based upon the above results, we reasoned that this invasive capacity might be dependent upon the active Dlg/SGEF pool that is maintained by E6/E7 expression. To investigate this we proceeded to perform a series of matrigel invasion assays using HeLa cells in which either E6/E7, hDlg or SGEF expression was ablated by siRNA transfection. Cells were transfected with the relevant siRNAs and after 72 hrs the cells were trypsinised and counted, and equal numbers of cells were inoculated into matrigel invasion chambers. At the same time, the efficiency of the siRNAs was determined by western blotting for the relevant target protein (Figure 9A). Chambers were left overnight at 37°C and the number of cells invading the lower chamber was counted the following day. The collated results from multiple assays are also shown in Figure 9A. Not surprisingly, loss of E6/E7 expression results in a dramatic decrease in the invasive capacity of these cells. However, loss of either hDlg or SGEF also results in a significant decrease in the capacity of these cells to invade the matrigel. We also repeated this assay in HPV-16 positive CaSki cells and the results shown in Figure 9B also confirm that continued expression of hDlg and SGEF contribute directly to the invasive potential of these cells. These results demonstrate that, in the context of HPV-induced malignancy, the residual levels of hDlg found within HPV-18 positive HeLa cells and HPV-16 positive CaSki cells actually contribute directly to the invasive potential of these cells, most likely through up-regulation of SGEF and RhoG activity.

Bottom Line: We also show that HPV-18 E6 can interact indirectly with SGEF in a manner that is dependent upon the presence of hDlg and PDZ binding capacity.In HPV transformed cells, E6 maintains a high level of RhoG activity, and this is dependent upon the presence of hDlg and SGEF, which are found in complex with E6.Furthermore, we show that E6, hDlg and SGEF each directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.

ABSTRACT
A major target of the HPV E6 oncoprotein is the human Discs Large (hDlg) tumour suppressor, although how this interaction contributes to HPV-induced malignancy is still unclear. Using a proteomic approach we show that a strong interacting partner of hDlg is the RhoG-specific guanine nucleotide exchange factor SGEF. The interaction between hDlg1 and SGEF involves both PDZ and SH3 domain recognition, and directly contributes to the regulation of SGEF's cellular localization and activity. Consistent with this, hDlg is a strong enhancer of RhoG activity, which occurs in an SGEF-dependent manner. We also show that HPV-18 E6 can interact indirectly with SGEF in a manner that is dependent upon the presence of hDlg and PDZ binding capacity. In HPV transformed cells, E6 maintains a high level of RhoG activity, and this is dependent upon the presence of hDlg and SGEF, which are found in complex with E6. Furthermore, we show that E6, hDlg and SGEF each directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells. These studies demonstrate that hDlg has a distinct oncogenic function in the context of HPV induced malignancy, one of the outcomes of which is increased RhoG activity and increased invasive capacity.

Show MeSH
Related in: MedlinePlus