Data documento
06/05/2024

Título
Comprehensive Molecular Landscape of Cetuximab Resistance in Head and Neck Cancer Cell Lines

Autor(es)
Gomes I.N.F. - da Silva-Oliveira R.J. - da Silva L.S. - Martinho O. - Evangelista A.F. - van Helvoort Lengert A. - Leal L.F. - Silva V.A.O. - Dos Santos S.P. - Nascimento F.C. - Lopes Carvalho A. - Reis R.M.

Palavras chave
EGFR - biomarkers - cetuximab - drug resistance - head and neck tumors - in vitro - pre-clinical

Resumo
Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in vitro model. We established a cetuximab resistant model (FaDu), using increased cetuximab concentrations for more than eight months. The resistance and parental cells were evaluated for cell viability and functional assays. Protein expression was analyzed by Western blot and human cell surface panel by lyoplate. The mutational profile and copy number alterations (CNA) were analyzed using whole-exome sequencing (WES) and the NanoString platform. FaDu resistant clones exhibited at least two-fold higher IC50 compared to the parental cell line. WES showed relevant mutations in several cancer-related genes, and the comparative mRNA expression analysis showed 36 differentially expressed genes associated with EGFR tyrosine kinase inhibitors resistance, RAS, MAPK, and mTOR signaling. Importantly, we observed that overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance. Protein analysis revealed EGFR phosphorylation inhibition and mTOR increase in resistant cells. Moreover, the resistant cell line demonstrated an aggressive phenotype with a significant increase in adhesion, the number of colonies, and migration rates. Overall, we identified several molecular alterations in the cetuximab resistant cell line that may constitute novel biomarkers of cetuximab response such as mTOR and RhoA overexpression. These findings indicate new strategies to overcome anti-EGFR resistance in HNSCC.

Citação
Gomes INF, da Silva-Oliveira RJ, da Silva LS, Martinho O, Evangelista AF, van Helvoort Lengert A, Leal LF, Silva VAO, Dos Santos SP, Nascimento FC, Lopes Carvalho A, Reis RM. Comprehensive Molecular Landscape of Cetuximab Resistance in Head and Neck Cancer Cell Lines. Cells. 2022 Jan 4;11(1):154. doi: 10.3390/cells11010154. PMID: 35011716; PMCID: PMC8750399.

Data documento
06/05/2024

Título
Digital expression profile of immune checkpoint genes in medulloblastomas identifies CD24 and CD276 as putative immunotherapy targets

Autor(es)
Marques R.F. - Moreno D.A. - da Silva L. - Leal L.F. - de Paula F.E. - Santana I. - Teixeira G. - Saggioro F. - Neder L. - Junior C.A. - Mançano B. - Reis R.M.

Palavras chave
immune checkpoints - immune profile - immunotherapy - medulloblastoma - nCounter mRNA expression assay

Resumo
Introduction: Medulloblastoma is the most common and lethal pediatric malignant brain tumor. It comprises four main molecular subgroups: WNT-activated, SHH-activated, Group 3, and Group 4. Medulloblastoma treatment is surgical resection, craniospinal radiation, and chemotherapy. However, many patients do not respond to therapy, and most suffer severe side effects. Cancer immunotherapy targeting immune checkpoints (IC) (PD-1, PD-L1, and CTLA4) has been getting disappointing outcomes in brain tumors. Nevertheless, other less explored immune checkpoints may be promising candidates for medulloblastoma therapy. Objectives: In the present study, we aimed to characterize the expression profile of 19 immune checkpoints in medulloblastoma. Methods: We analyzed 88 formalin-fixed paraffin-embedded medulloblastomas previously classified for each molecular subgroup and three non-tumoral brain tissue. mRNA levels of 19 immune checkpoint-related genes were quantified using the nCounter (PanCancer Immune Profiling Panel) assay. Further in silico analysis was performed in two larger public microarray datasets, one of which enabled comparisons between tumoral and non-tumoral tissues. Immunohistochemistry of PD-L1 was performed in a subset of cases. Microsatellite instability was also molecularly analyzed. Results: We observed an absence of expression of the canonic ICs, namely PDCD1 (PD-1), CD274 (PD-L1), and CTLA4, as well as CD80, CD86, BTLA, IDO1, CD48, TNFSF14, CD160, CEACAM1, and CD244. PD-L1 protein expression was also practically absent. We found higher mRNA levels of CD24, CD47, CD276 (B7-H3), and PVR, and lower mRNA levels of HAVCR2, LAG3, and TIGIT genes, with significant differences across the four molecular subgroups. Compared to the non-tumor tissues, the expression levels of CD276 in all subgroups and CD24 in SHH, Group 3, and Group 4 subgroups are significantly higher. The in silico analysis confirmed the expression profile found in the Brazilian cohort, including the lower/absent expression of the canonic ICs. Moreover, it confirmed the overexpression of CD24 and CD276 in medulloblastomas compared with the non-tumor tissue. Additionally, CD276 and CD24 high levels were associated with worse survival. Conclusion: These results highlight the low or absence of mRNA levels of the canonic targetable ICs in medulloblastomas. Importantly, the analysis revealed overexpression of CD24 and CD276, which can constitute prognostic biomarkers and attractive immunotherapy targets for medulloblastomas.

Citação
Marques RF, Moreno DA, da Silva L, Leal LF, de Paula FE, Santana I, Teixeira G, Saggioro F, Neder L, Junior CA, Mançano B, Reis RM. Digital expression profile of immune checkpoint genes in medulloblastomas identifies CD24 and CD276 as putative immunotherapy targets. Front Immunol. 2023 Feb 7;14:1062856. doi: 10.3389/fimmu.2023.1062856. PMID: 36825029; PMCID: PMC9941636.