TY - JOUR
T1 - CXCR4-targeted nanotoxins induce GSDME-dependent pyroptosis in head and neck squamous cell carcinoma
AU - Rioja-Blanco, Elisa
AU - Arroyo-Solera, Irene
AU - Álamo, Patricia
AU - Casanova, Isolda
AU - Gallardo, Alberto
AU - Unzueta, Ugutz
AU - Serna, Naroa
AU - Sánchez-García, Laura
AU - Quer, Miquel
AU - Villaverde, Antonio
AU - Vázquez, Esther
AU - León, Xavier
AU - Alba-Castellón, Lorena
AU - Mangues, Ramon
N1 - Funding Information:
The authors would like to thank Clara Seira (IIB-Sant Pau, Barcelona) for her technical support, and Dr. Gemma Vilahur (IIB-Sant Pau, Barcelona) for letting us use the Mindray BC-5000 Vet hematology analyzer.
Funding Information:
This work was supported by grants PI21/00150, PI18/ 00650, PIE15/00028, PI15/00378 and EU COST Action CA 17140 to Ramon Mangues, PI19/01661 to Xavier León, and PI17/00584 to Miquel Quer, founded by Instituto de Salud Carlos III (ISCIII, Co-funding from FEDER, Spain). Grants BIO2016–76063-R, AEI/FEDER, UE to Antonio Villaverde and PID2019-105416RB-I00/AEI/ https://doi.org/10.13039/501100011033 to Esther Vázquez, founded by Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (Spain). CIBER-BBN (Spain) grants CB06/01/ 1031 and 4NanoMets to Ramon Mangues, VENOM4CANCER to Antonio Villaverde, NANOREMOTE to Esther Vázquez, and NANOSCAPE to Ugutz Unzueta. AGAUR 2017- SGR-865 (Spain) to Ramon Mangues, and 2017SGR-229 (Spain) to Antonio Villaverde. Josep Carreras Leukemia Research Institute (Spain) P/AG to Ramon Mangues. Elisa Rioja-Blanco and Laura Sánchez-García were supported by a predoctoral fellowship from AGAUR (2020FI_B2 00168 and 2018FI_B2_00051 (Spain)) co-funded by European Social Fund (ESF investing in your future). Lorena Alba-Castellón was supported by a postdoctoral fellowship from AECC (Spanish Association of Cancer Research, Spain). Antonio Villaverde was granted an Icrea Academia Award (Spain). Ugutz Unzueta was also supported by Grant PERIS SLT006/17/00093 from la Generalitat de Catalunya (Spain) and Miguel Servet fellowship (CP19/00028) from Instituto de Salud Carlos III (Spain) co-funded by European Social Fund (ESF investing in your future). The toxicity studies have been performed in the ICTS-141007 Nanbiosis Platform, using its CIBER-BBN Nanotoxicology Unit ( http://www.nanbiosis.es/portfolio/u18-nanotoxicology-unit/ ). Protein production has been partially performed by the ICTS “NANBIOSIS”, more specifically by the Protein Production Platform of CIBER-BBN/ IBB ( http://www.nanbiosis.es/unit/u1-protein-production-platform-ppp/ ).
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/2/4
Y1 - 2022/2/4
N2 - Background: Therapy resistance, which leads to the development of loco-regional relapses and distant metastases after treatment, constitutes one of the major problems that head and neck squamous cell carcinoma (HNSCC) patients currently face. Thus, novel therapeutic strategies are urgently needed. Targeted drug delivery to the chemokine receptor 4 (CXCR4) represents a promising approach for HNSCC management. In this context, we have developed the self-assembling protein nanotoxins T22-PE24-H6 and T22-DITOX-H6, which incorporate the de-immunized catalytic domain of Pseudomonas aeruginosa (PE24) exotoxin A and the diphtheria exotoxin (DITOX) domain, respectively. Both nanotoxins contain the T22 peptide ligand to specifically target CXCR4-overexpressing HNSCC cells. In this study, we evaluate the potential use of T22-PE24-H6 and T22-DITOX-H6 nanotoxins for the treatment of HNSCC. Methods: T22-PE24-H6 and T22-DITOX-H6 CXCR4-dependent cytotoxic effect was evaluated in vitro in two different HNSCC cell lines. Both nanotoxins cell death mechanisms were assessed in HNSCC cell lines by phase-contrast microscopy, AnnexinV/ propidium iodide (PI) staining, lactate dehydrogenase (LDH) release assays, and western blotting. Nanotoxins antitumor effect in vivo was studied in a CXCR4+ HNSCC subcutaneous mouse model. Immunohistochemistry, histopathology, and toxicity analyses were used to evaluate both nanotoxins antitumor effect and possible treatment toxicity. GSMDE and CXCR4 expression in HNSCC patient tumor samples was also assessed by immunohistochemical staining. Results: First, we found that both nanotoxins exhibit a potent CXCR4-dependent cytotoxic effect in vitro. Importantly, nanotoxin treatment triggered caspase-3/Gasdermin E (GSDME)-mediated pyroptosis. The activation of this alternative cell death pathway that differs from traditional apoptosis, becomes a promising strategy to bypass therapy resistance. In addition, T22-PE24-H6 and T22-DITOX-H6 displayed a potent antitumor effect in the absence of systemic toxicity in a CXCR4+ subcutaneous HNSCC mouse model. Lastly, GSDME was found to be overexpressed in tumor tissue from HNSCC patients, highlighting the relevance of this strategy. Conclusions: Altogether, our results show that T22-PE24-H6 and T22-DITOX-H6 represent a promising therapy for HNSCC patients. Remarkably, this is the first study showing that both nanotoxins are capable of activating caspase-3/GSDME-dependent pyroptosis, opening a novel avenue for HNSCC treatment.
AB - Background: Therapy resistance, which leads to the development of loco-regional relapses and distant metastases after treatment, constitutes one of the major problems that head and neck squamous cell carcinoma (HNSCC) patients currently face. Thus, novel therapeutic strategies are urgently needed. Targeted drug delivery to the chemokine receptor 4 (CXCR4) represents a promising approach for HNSCC management. In this context, we have developed the self-assembling protein nanotoxins T22-PE24-H6 and T22-DITOX-H6, which incorporate the de-immunized catalytic domain of Pseudomonas aeruginosa (PE24) exotoxin A and the diphtheria exotoxin (DITOX) domain, respectively. Both nanotoxins contain the T22 peptide ligand to specifically target CXCR4-overexpressing HNSCC cells. In this study, we evaluate the potential use of T22-PE24-H6 and T22-DITOX-H6 nanotoxins for the treatment of HNSCC. Methods: T22-PE24-H6 and T22-DITOX-H6 CXCR4-dependent cytotoxic effect was evaluated in vitro in two different HNSCC cell lines. Both nanotoxins cell death mechanisms were assessed in HNSCC cell lines by phase-contrast microscopy, AnnexinV/ propidium iodide (PI) staining, lactate dehydrogenase (LDH) release assays, and western blotting. Nanotoxins antitumor effect in vivo was studied in a CXCR4+ HNSCC subcutaneous mouse model. Immunohistochemistry, histopathology, and toxicity analyses were used to evaluate both nanotoxins antitumor effect and possible treatment toxicity. GSMDE and CXCR4 expression in HNSCC patient tumor samples was also assessed by immunohistochemical staining. Results: First, we found that both nanotoxins exhibit a potent CXCR4-dependent cytotoxic effect in vitro. Importantly, nanotoxin treatment triggered caspase-3/Gasdermin E (GSDME)-mediated pyroptosis. The activation of this alternative cell death pathway that differs from traditional apoptosis, becomes a promising strategy to bypass therapy resistance. In addition, T22-PE24-H6 and T22-DITOX-H6 displayed a potent antitumor effect in the absence of systemic toxicity in a CXCR4+ subcutaneous HNSCC mouse model. Lastly, GSDME was found to be overexpressed in tumor tissue from HNSCC patients, highlighting the relevance of this strategy. Conclusions: Altogether, our results show that T22-PE24-H6 and T22-DITOX-H6 represent a promising therapy for HNSCC patients. Remarkably, this is the first study showing that both nanotoxins are capable of activating caspase-3/GSDME-dependent pyroptosis, opening a novel avenue for HNSCC treatment.
KW - CXCR4
KW - GSDME
KW - HNSCC
KW - Pyroptosis
KW - Targeted drug delivery
UR - http://www.scopus.com/inward/record.url?scp=85124175207&partnerID=8YFLogxK
U2 - https://doi.org/10.1186/s13046-022-02267-8
DO - https://doi.org/10.1186/s13046-022-02267-8
M3 - Article
C2 - 35120582
AN - SCOPUS:85124175207
SN - 0392-9078
VL - 41
JO - Journal of Experimental and Clinical Cancer Research
JF - Journal of Experimental and Clinical Cancer Research
IS - 1
M1 - 49
ER -