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Prolonged cetuximab treatment promotes p27^Kip1-mediated G1 arrest and autophagy in head and neck squamous cell carcinoma.

ABSTRACT: Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is an efficient anti-tumor therapeutic agent that inhibits the activation of EGFR; however, data related to the cellular effects of prolonged cetuximab treatment are limited. In this study, the long-term cellular outcome of prolonged cetuximab treatment and the related molecular mechanism were explored in a head and neck squamous cell carcinoma cell line constitutively expressing a fluorescent ubiquitination-based cell cycle indicator. Fluorescent time-lapse imaging was used to assess clonal growth, cell motility, and cell-cycle progression. Western blot analysis was performed to measure the level of phosphorylation and protein-expression following cetuximab treatment. Over 5 days cetuximab treatment decreased cell motility and enhanced G1 phase cell arrest in the central region of the colonies. Significantly decreased phosphorylation of retinoblastoma, Skp2, and Akt-mTOR proteins, accumulation of p27^Kip1, and induction of type II LC3B were observed over 8 days cetuximab treatment. Results of the present study elucidate the cetuximab-dependent inhibition of cell migration, resulting in high cell density-related stress and persistent cell-cycle arrest at G1 phase culminating in autophagy. These findings provide novel molecular insights related to the anti-tumor effects of prolonged cetuximab treatment with the potential to improve future therapeutic strategy.

SUBMITTER: Okuyama K

PROVIDER: S-EPMC7933308 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Prolonged cetuximab treatment promotes p27<sup>Kip1</sup>-mediated G1 arrest and autophagy in head and neck squamous cell carcinoma.

Okuyama Kohei K Suzuki Keiji K Naruse Tomofumi T Tsuchihashi Hiroki H Yanamoto Souichi S Kaida Atsushi A Miura Masahiko M Umeda Masahiro M Yamashita Shunichi S

Scientific reports 20210304 1

Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is an efficient anti-tumor therapeutic agent that inhibits the activation of EGFR; however, data related to the cellular effects of prolonged cetuximab treatment are limited. In this study, the long-term cellular outcome of prolonged cetuximab treatment and the related molecular mechanism were explored in a head and neck squamous cell carcinoma cell line constitutively expressing a fluorescent ubiquitination-based ce ...[more]

PMID: 33664437

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Project description:To show the relationship between antibody delivery and therapeutic efficacy in head and neck cancers, in this study we evaluated the pharmacokinetics and pharmacodynamics of epidermal growth factor receptor (EGFR)-targeted immunotherapy and radioimmunotherapy by quantitative positron emission tomography (PET) imaging.EGFR expression on UM-SCC-22B and SCC1 human head and neck squamous cell cancer (HNSCC) cells were determined by flow cytometry and immunostaining. Tumor delivery and distribution of cetuximab in tumor-bearing nude mice were evaluated with small animal PET using (64)Cu-DOTA-cetuximab. The in vitro toxicity of cetuximab to HNSCC cells was evaluated by MTT assay. The tumor-bearing mice were then treated with four doses of cetuximab at 10 mg/kg per dose, and tumor growth was evaluated by caliper measurement. FDG PET was done after the third dose of antibody administration to evaluate tumor response. Apoptosis and tumor cell proliferation after cetuximab treatment were analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Ki-67 staining. Radioimmunotherapy was done with (90)Y-DOTA-cetuximab.EGFR expression on UM-SCC-22B cells is lower than that on SCC1 cells. However, the UM-SCC-22B tumors showed much higher (64)Cu-DOTA-cetuximab accumulation than the SCC1 tumors. Cetuximab-induced apoptosis in SCC1 tumors and tumor growth was significantly inhibited, whereas an agonistic effect of cetuximab on UM-SCC-22B tumor growth was observed. After cetuximab treatment, the SCC1 tumors showed decreased FDG uptake, and the UM-SCC-22B tumors had increased FDG uptake. UM-SCC-22B tumors are more responsive to (90)Y-DOTA-cetuximab treatment than SCC1 tumors, partially due to the high tumor accumulation of the injected antibody.Cetuximab has an agonistic effect on the growth of UM-SCC-22B tumors, indicating that tumor response to cetuximab treatment is not necessarily related to EGFR expression and antibody delivery efficiency, as determined by PET imaging. Although PET imaging with antibodies as tracers has limited function in patient screening, it can provide guidance for targeted therapy using antibodies as delivery vehicles.

Project description:Background & aimsOncogenic mutations in KRAS, coupled with inactivation of p53, CDKN2A/p16INK4A, and SMAD4, drive progression of pancreatic ductal adenocarcinoma (PDA). Overexpression of MYC and deregulation of retinoblastoma (RB) further promote cell proliferation and make identifying a means to therapeutically alter cell-cycle control pathways in PDA a significant challenge. We previously showed that the basic helix-loop-helix transcription factor E47 induced stable growth arrest in PDA cells in vitro and in vivo. Here, we identified molecular mechanisms that underlie E47-induced growth arrest in low-passage, patient-derived primary and established PDA cell lines.MethodsRNA sequencing was used to profile E47-dependent transcriptomes in 5 PDA cell lines. Gene Ontology analysis identified cell-cycle control as the most altered pathway. Small interfering RNA/short hairpin RNA knockdown, small-molecule inhibitors, and viral expression were used to examine the function of E47-dependent genes in cell-cycle arrest. Cell morphology, expression of molecular markers, and senescence-associated β-galactosidase activity assays identified cellular senescence.ResultsE47 uniformly inhibited PDA cell-cycle progression by decreasing expression of MYC, increasing the level of CDKN1B/p27KIP1, and restoring RB tumor-suppressor function. The molecular mechanisms by which E47 elicited these changes included altering both RNA transcript levels and protein stability of MYC and CDKN1B/p27KIP1. At the cellular level, E47 elicited a senescence-like phenotype characterized by increased senescence-associated β-galactosidase activity and altered expression of senescence markers.ConclusionsE47 governs a highly conserved network of cell-cycle control genes, including MYC, CDKN1B/p27KIP1, and RB, which can induce a senescence-like program in PDA cells that lack CDKN2A/p16INK4A and wild-type p53. RNA sequencing data are available at the National Center for Biotechnology Information GEO at https://www.ncbi.nlm.nih.gov/geo/; accession number: GSE100327.

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Prolonged cetuximab treatment promotes p27^Kip1-mediated G1 arrest and autophagy in head and neck squamous cell carcinoma.

Publications

Prolonged cetuximab treatment promotes p27<sup>Kip1</sup>-mediated G1 arrest and autophagy in head and neck squamous cell carcinoma.

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Prolonged cetuximab treatment promotes p27Kip1-mediated G1 arrest and autophagy in head and neck squamous cell carcinoma.

Publications

Prolonged cetuximab treatment promotes p27<sup>Kip1</sup>-mediated G1 arrest and autophagy in head and neck squamous cell carcinoma.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

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Prolonged cetuximab treatment promotes p27^Kip1-mediated G1 arrest and autophagy in head and neck squamous cell carcinoma.