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RIPK3 expression in cervical cancer cells is required for PolyIC-induced necroptosis, IL-1α release, and efficient paracrine dendritic cell activation.

Schmidt SV, Seibert S, Walch-Rückheim B, Vicinus B, Kamionka EM, Pahne-Zeppenfeld J, Solomayer EF, Kim YJ, Bohle RM, Smola S - Oncotarget (2015)

Bottom Line: However, little is known about the molecular requirements resulting in successful immune activation.Again both, IL-1α release and DC activation, were strictly dependent on RIPK3 expression in the tumor cells.Of note, our in situ analyses revealed heterogeneous RIPK3 expression patterns in cervical squamous cell carcinomas and adenocarcinomas.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine Cologne and Institute of Virology, University of Cologne, Germany.

ABSTRACT
Previous studies have shown that cervical cancer cells only release low levels of pro-inflammatory cytokines owing to infection with human papillomaviruses. This results in low immunogenicity of the cancer cells. The viral dsRNA analog PolyIC has been suggested as a promising adjuvant for cervical cancer immunotherapy. However, little is known about the molecular requirements resulting in successful immune activation. Here, we demonstrate that stimulation of cervical cancer cells with PolyIC induced necroptotic cell death, which was strictly dependent on the expression of the receptor-interacting protein kinase RIPK3. Necroptotic cancer cells released interleukin-1α (IL-1α), which was required for powerful activation of dendritic cells (DC) to produce IL-12, a cytokine critical for anti-tumor responses. Again both, IL-1α release and DC activation, were strictly dependent on RIPK3 expression in the tumor cells. Of note, our in situ analyses revealed heterogeneous RIPK3 expression patterns in cervical squamous cell carcinomas and adenocarcinomas. In summary, our study identified a novel RIPK3-dependent mechanism that explains how PolyIC-treatment of cervical cancer cells leads to potent DC activation. Our findings suggest that the RIPK3 expression status in cervical cancer cells might critically influence the outcome of PolyIC-based immunotherapeutic approaches and should therefore be assessed prior to immunotherapy.

No MeSH data available.


Related in: MedlinePlus

HMGB1 is released from PolyIC-stimulated C4-I cells(A) C4-I and HeLa cells were pre-incubated with Z-VAD for 30 min and stimulated with medium or PolyIC for 24 h. Equal amounts of supernatant were analyzed for HMGB1 release in Western blot using HMGB1-specific antibodies. (B) HMGB1 does not enhance PolyIC-induced IL-12 expression. C4-I cells were stimulated with medium or PolyIC in the absence (black bars) or presence (grey bars) of rhHMGB1. PolyIC-induced IL-12 expression in DC was set at 100%. The mean values ± SD from n = 2 experiments performed in duplicate are shown. (C) DC were stimulated with medium or supernatant from PolyIC-treated C4-I cells in the absence or presence of RAGE/Fc. The IL-12 expression induced by supernatant from PolyIC-treated C4-I cells was set at 100%. The mean values ± SD of n = 3 experiments performed in duplicate are shown.
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Figure 5: HMGB1 is released from PolyIC-stimulated C4-I cells(A) C4-I and HeLa cells were pre-incubated with Z-VAD for 30 min and stimulated with medium or PolyIC for 24 h. Equal amounts of supernatant were analyzed for HMGB1 release in Western blot using HMGB1-specific antibodies. (B) HMGB1 does not enhance PolyIC-induced IL-12 expression. C4-I cells were stimulated with medium or PolyIC in the absence (black bars) or presence (grey bars) of rhHMGB1. PolyIC-induced IL-12 expression in DC was set at 100%. The mean values ± SD from n = 2 experiments performed in duplicate are shown. (C) DC were stimulated with medium or supernatant from PolyIC-treated C4-I cells in the absence or presence of RAGE/Fc. The IL-12 expression induced by supernatant from PolyIC-treated C4-I cells was set at 100%. The mean values ± SD of n = 3 experiments performed in duplicate are shown.

Mentions: We were interested in determining which factor released during PolyIC-mediated necroptosis was responsible for the enhancement of DC activation. HMGB1, an alarmin with immunostimulatory capacity that is passively released during necrosis [42], was found in supernatants from PolyIC-stimulated C4-I cells, but not in supernatants from HeLa cells. Z-VAD did not affect HMGB1 release (Figure 5A). A loading control for this experiment is shown in Supplementary Figure S2. Recombinant HMGB1 did not directly activate DC or enhance the effect of PolyIC (Figure 5B). Naturally expressed HMGB1 may be post-translationally modified, and may differ from bacterially expressed HMGB1 with respect to functional activity. Therefore, supernatants from PolyIC-activated C4-I cells were neutralized using the soluble receptor construct RAGE/Fc [43]. RAGE/Fc did not significantly interfere with the IL-12 production induced by supernatants from PolyIC-stimulated C4-I cells (Figure 5C). These data indicated that HMGB1 was released during PolyIC-mediated necroptosis, but was not responsible for enhanced IL-12 production in DC.


RIPK3 expression in cervical cancer cells is required for PolyIC-induced necroptosis, IL-1α release, and efficient paracrine dendritic cell activation.

Schmidt SV, Seibert S, Walch-Rückheim B, Vicinus B, Kamionka EM, Pahne-Zeppenfeld J, Solomayer EF, Kim YJ, Bohle RM, Smola S - Oncotarget (2015)

HMGB1 is released from PolyIC-stimulated C4-I cells(A) C4-I and HeLa cells were pre-incubated with Z-VAD for 30 min and stimulated with medium or PolyIC for 24 h. Equal amounts of supernatant were analyzed for HMGB1 release in Western blot using HMGB1-specific antibodies. (B) HMGB1 does not enhance PolyIC-induced IL-12 expression. C4-I cells were stimulated with medium or PolyIC in the absence (black bars) or presence (grey bars) of rhHMGB1. PolyIC-induced IL-12 expression in DC was set at 100%. The mean values ± SD from n = 2 experiments performed in duplicate are shown. (C) DC were stimulated with medium or supernatant from PolyIC-treated C4-I cells in the absence or presence of RAGE/Fc. The IL-12 expression induced by supernatant from PolyIC-treated C4-I cells was set at 100%. The mean values ± SD of n = 3 experiments performed in duplicate are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496172&req=5

Figure 5: HMGB1 is released from PolyIC-stimulated C4-I cells(A) C4-I and HeLa cells were pre-incubated with Z-VAD for 30 min and stimulated with medium or PolyIC for 24 h. Equal amounts of supernatant were analyzed for HMGB1 release in Western blot using HMGB1-specific antibodies. (B) HMGB1 does not enhance PolyIC-induced IL-12 expression. C4-I cells were stimulated with medium or PolyIC in the absence (black bars) or presence (grey bars) of rhHMGB1. PolyIC-induced IL-12 expression in DC was set at 100%. The mean values ± SD from n = 2 experiments performed in duplicate are shown. (C) DC were stimulated with medium or supernatant from PolyIC-treated C4-I cells in the absence or presence of RAGE/Fc. The IL-12 expression induced by supernatant from PolyIC-treated C4-I cells was set at 100%. The mean values ± SD of n = 3 experiments performed in duplicate are shown.
Mentions: We were interested in determining which factor released during PolyIC-mediated necroptosis was responsible for the enhancement of DC activation. HMGB1, an alarmin with immunostimulatory capacity that is passively released during necrosis [42], was found in supernatants from PolyIC-stimulated C4-I cells, but not in supernatants from HeLa cells. Z-VAD did not affect HMGB1 release (Figure 5A). A loading control for this experiment is shown in Supplementary Figure S2. Recombinant HMGB1 did not directly activate DC or enhance the effect of PolyIC (Figure 5B). Naturally expressed HMGB1 may be post-translationally modified, and may differ from bacterially expressed HMGB1 with respect to functional activity. Therefore, supernatants from PolyIC-activated C4-I cells were neutralized using the soluble receptor construct RAGE/Fc [43]. RAGE/Fc did not significantly interfere with the IL-12 production induced by supernatants from PolyIC-stimulated C4-I cells (Figure 5C). These data indicated that HMGB1 was released during PolyIC-mediated necroptosis, but was not responsible for enhanced IL-12 production in DC.

Bottom Line: However, little is known about the molecular requirements resulting in successful immune activation.Again both, IL-1α release and DC activation, were strictly dependent on RIPK3 expression in the tumor cells.Of note, our in situ analyses revealed heterogeneous RIPK3 expression patterns in cervical squamous cell carcinomas and adenocarcinomas.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine Cologne and Institute of Virology, University of Cologne, Germany.

ABSTRACT
Previous studies have shown that cervical cancer cells only release low levels of pro-inflammatory cytokines owing to infection with human papillomaviruses. This results in low immunogenicity of the cancer cells. The viral dsRNA analog PolyIC has been suggested as a promising adjuvant for cervical cancer immunotherapy. However, little is known about the molecular requirements resulting in successful immune activation. Here, we demonstrate that stimulation of cervical cancer cells with PolyIC induced necroptotic cell death, which was strictly dependent on the expression of the receptor-interacting protein kinase RIPK3. Necroptotic cancer cells released interleukin-1α (IL-1α), which was required for powerful activation of dendritic cells (DC) to produce IL-12, a cytokine critical for anti-tumor responses. Again both, IL-1α release and DC activation, were strictly dependent on RIPK3 expression in the tumor cells. Of note, our in situ analyses revealed heterogeneous RIPK3 expression patterns in cervical squamous cell carcinomas and adenocarcinomas. In summary, our study identified a novel RIPK3-dependent mechanism that explains how PolyIC-treatment of cervical cancer cells leads to potent DC activation. Our findings suggest that the RIPK3 expression status in cervical cancer cells might critically influence the outcome of PolyIC-based immunotherapeutic approaches and should therefore be assessed prior to immunotherapy.

No MeSH data available.


Related in: MedlinePlus