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EGFR-targeted mAb therapy modulates autophagy in head and neck squamous cell carcinoma through NLRX1-TUFM protein complex.

ABSTRACT: Epidermal growth factor receptor (EGFR)-targeted therapy in head and neck squamous cell carcinoma (HNSCC) patients frequently results in tumor resistance to treatment. Autophagy is an emerging underlying resistance mechanism, however, the molecular autophagy machinery in HNSCC cells and potential biomarkers of patient response to EGFR-targeted therapy remain insufficiently characterized. Here we show that the EGFR blocking with cetuximab leads to varied autophagic responses, which modulate cancer cell susceptibility to EGFR inhibition. Inhibition of autophagy sensitizes HNSCC cells to EGFR blockade. Importantly, we identify a novel signaling hub centering on the NLRX1 (nucleotide-binding, lots of leucine-rich repeats-containing protein member X1)-TUFM (Tu translation elongation factor mitochondrial) protein complex, promoting autophagic flux. Defects in the expression of either NLRX1 or TUFM result in compromised autophagy when treated with EGFR inhibitors. As a previously undefined autophagy-promoting mechanism, we found that TUFM serves as a novel anchorage site, recruiting Beclin-1 to mitochondria, promoting its polyubiquitination, and interfering with its interaction with Rubicon. This protein complex is also essential for endoplasmic reticulum stress signaling induction, possibly as an additional mechanism to promote autophagy. Utilizing tumor specimens from a novel neoadjuvant clinical trial, we show that increased expression of the autophagy adaptor protein, SQSTM1/p62, is associated with poor response to cetuximab therapy. These findings expand our understanding of the components involved in HNSCC autophagy machinery that responds to EGFR inhibitors, and suggest potential combinatorial approaches to enhance its therapeutic efficacy.

SUBMITTER: Lei Y

PROVIDER: S-EPMC5257174 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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EGFR-targeted mAb therapy modulates autophagy in head and neck squamous cell carcinoma through NLRX1-TUFM protein complex.

Lei Y Y Kansy B A BA Li J J Cong L L Liu Y Y Trivedi S S Wen H H Ting J P-Y JP Ouyang H H Ferris R L RL

Oncogene 20160215 36

Epidermal growth factor receptor (EGFR)-targeted therapy in head and neck squamous cell carcinoma (HNSCC) patients frequently results in tumor resistance to treatment. Autophagy is an emerging underlying resistance mechanism, however, the molecular autophagy machinery in HNSCC cells and potential biomarkers of patient response to EGFR-targeted therapy remain insufficiently characterized. Here we show that the EGFR blocking with cetuximab leads to varied autophagic responses, which modulate cance ...[more]

PMID: 26876213

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Project description:In head and neck squamous cell cancer, the human epidermal growth factor receptor 1 (EGFR) is the dominant signaling molecule among all members of the family. So far, cetuximab is the only approved anti-EGFR monoclonal antibody used for the treatment of head and neck squamous cell cancer, but despite the benefits of adding it to standard treatment regimens, attempts to define a predictive biomarker to stratify patients for cetuximab treatment have been unsuccessful. We hypothesized that imaging with EGFR-specific radioligands may facilitate noninvasive measurement of EGFR expression across the entire tumor burden and allow for dynamic monitoring of cetuximab-mediated changes in receptor expression. Methods: EGFR-specific Affibody molecule (ZEGFR:03115) was radiolabeled with 89Zr and 18F. The radioligands were characterized in vitro and in mice bearing subcutaneous tumors with varying levels of EGFR expression. The protein dose for imaging studies was assessed by injecting 89Zr-deferoxamine-ZEGFR:03115 (2.4-3.6 MBq, 2 μg) either together with or 30 min after increasing amounts of unlabeled ZEGFR:03115 (1, 5, 10, 15, and 20 μg). PET images were acquired at 3, 24, and 48 h after injection, and the image quantification data were correlated with the biodistribution results. The EGFR expression and biodistribution of the tracer were assessed ex vivo by immunohistochemistry, Western blot, and autoradiography. To downregulate the EGFR level, treatment with cetuximab was performed, and 18F-aluminium fluoride-NOTA-ZEGFR:03115 (12 μg, 1.5-2 MBq/mouse) was used to monitor receptor changes. Results: In vivo studies demonstrated that coinjecting 10 μg of nonlabeled molecules with 89Zr-deferoxamine-ZEGFR:03115 allows for clear tumor visualization 3 h after injection. The radioconjugate tumor accumulation was EGFR-specific, and PET imaging data showed a clear differentiation between xenografts with varying EGFR expression levels. A strong correlation was observed between PET analysis, ex vivo estimates of tracer concentration, and receptor expression in tumor tissues. Additionally, 18F-aluminium fluoride-NOTA-ZEGFR:03115 could measure receptor downregulation in response to EGFR inhibition. Conclusion: ZEGFR:03115-based radioconjugates can assess different levels of EGFR level in vivo and measure receptor expression changes in response to cetuximab, indicating a potential for assessment of adequate treatment dosing with anti-EGFR antibodies.

Project description:UnlabelledEpidermal growth factor receptor (EGFR) is a well-characterized protooncogene that has been shown to promote tumor progression in solid cancers. Clinical results for EGFR targeting with specific monoclonal antibodies (mAbs) such as cetuximab and panitumumab are promising; however, most studies indicate that only a subgroup of patients receiving the mAbs benefit from the immunotherapy, independent of EGFR expression level. To understand the in vivo kinetics of antibody delivery and localization, we performed small-animal PET studies with (64)Cu-labeled panitumumab in xenografts derived from 3 cell lines of human head and neck squamous cell carcinoma (HNSCC).MethodsNude mice bearing HNSCC tumors with different levels of EGFR expression were imaged with small-animal PET using (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-panitumumab. Antibody distribution in the tumors was confirmed by ex vivo immunostaining using panitumumab and fluorescein 5(6)-isothiocyanate (FITC) panitumumab. CD31 immunostaining and Evans blue assay were also performed to assess the tumor vascular density and permeability.ResultsAmong these 3 tumor models, UM-SCC-22B tumors with the lowest EGFR protein expression showed the highest (64)Cu-DOTA-panitumumab accumulation, whereas SQB20 tumors with the highest EGFR expression showed the lowest (64)Cu-DOTA-panitumumab accumulation. Ex vivo staining demonstrated that SQB20 cells still had extremely high EGFR expression after forming tumors in nude mice, indicating that the low uptake of (64)Cu-DOTA-panitumumab in SQB20 tumors was not due to the loss of EGFR expression. The results from CD31 immunostaining and Evans blue permeability assay suggest that the low vessel density, poor vascular permeability, and binding site barrier are likely responsible for the overall low tumor uptake of the highly EGFR-expressing SQB20 tumors.ConclusionThe results from this study provide a possible explanation for the lack of an observed correlation between therapeutic efficacy of cetuximab and panitumumab and EGFR expression level as determined by immunohistochemistry or fluorescent in situ hybridization and may shed new light on the complications of anti-EGFR mAb therapy for HNSCC and other malignancies.

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EGFR-targeted mAb therapy modulates autophagy in head and neck squamous cell carcinoma through NLRX1-TUFM protein complex.

Publications

EGFR-targeted mAb therapy modulates autophagy in head and neck squamous cell carcinoma through NLRX1-TUFM protein complex.

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