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Background: Currently, human papillomavirus (HPV) positivity represents a strong prognostic factor for both reduced risk of relapse and improved survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). However, a subset of HPV-positive OPSCC patients still experience poor outcomes. Furthermore, HPV-negative OPSCC patients, who have an even higher risk of relapse, are still lacking suitable prognostic biomarkers for clinical outcome. Here, we evaluated the prognostic value of LINE-1 methylation level in OPSCC patients, and further addressed the relationship between LINE-1 methylation status and p53 protein expression as well as genome-wide/gene-specific DNA methylation. Results: In this study, DNA was extracted from 163 formalin-fixed paraffin-embedded tissue samples retrospectively collected from stage III-IVB OPSCC patients managed with curative intent with up-front treatment. Quantitative methylation-specific PCR revealed that LINE-1 hypomethylation was directly associated with poor prognosis (5-year overall survival - OS: 28.1% for LINE-1 methylation Conclusions: Evaluation of the methylation level of LINE-1 may help in identifying the subset of OPSCC patients with bad prognosis regardless of their HPV status. Aberrant LINE-1 hypomethylation might occur along with TP53 mutations, and lead to altered gene expression in OPSCC. Citation Format: Mariateresa Casarotto, Valentina Lupato, Giorgio Giurato, Roberto Guerrieri, Sandro Sulfaro, Annamaria Salvati, Elisa D'Angelo, Carlo Furlan, Anna Menegaldo, Lorena Baboci, Barbara Montico, Irene Turturici, Riccardo Dolcetti, Salvatore Romeo, Vittorio Baggio, Stefania Corrado, Gianluca Businello, Maria Guido, Alessandro Weisz, Vittorio Giacomarra, Giovanni Franchin, Agostino Steffan, Luca Sigalotti, Emanuela Vaccher, Paolo Boscolo-Rizzo, Jerry Polesel, Giuseppe Fanetti, Elisabetta Fratta. LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1005.

Introduction: At present, the precise role of Spry proteins in tumor progression is still debated. Our previous study showed that Spry1 knockdown (Spry1KO) in BRAFV600-mutant cutaneous melanoma (CM) promoted cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and reduced tumor growth in vivo. Furthermore, we observed that Spry1 loss impaired angiogenesis in vivo. Hypoxia has been recognized as one of the crucial features of CM. Hypoxia results in the activation of the transcription factor hypoxia-inducible factor 1α (HIF-1α), which represents a master regulator of angiogenesis. Hence, the present study was designed to explore a possible role of Spry1 in modulating angiogenesis and hypoxia in CM. Material and method: SPRY1 gene was knocked-out using the CRISPR-strategy in the BRAF wild-type (wt) Mel 313 and in the BRAF-mutant Mel 593 cell lines. Angiogenic potential was assessed through in vivo CD31 staining and in vitro tube formation assays. By using in vitro and in vivo models, the effects of Spry1KO on hypoxia-related genes were investigated through RNA-sequencing (RNA-seq), quantitative real-time PCR, western blot, and immunofluorescence analyses. To gain insight into Spry1 interactome, immunoprecipitation coupled to mass-spectrometry (IP-MS) was employed. Results and discussion: Consistent with our previous data, tumors arising from Mel 313 and Mel 593 Spry1KO clones were significantly smaller than those from their corresponding parental cells. Tumor growth inhibition was accompanied by a substantial reduction of angiogenesis, as assessed by the immunofluorescence staining of CD31 in vivo and the tube formation assay in vitro. Accordingly, RNA-seq analysis indicated that the vascular endothelial growth factor A was significantly down-regulated following Spry1 silencing. Notably, Ingenuity Pathway Analysis identified “HIF-1α Signaling” as the most significant molecular and cellular function affected in vivo by Spry1 silencing regardless of the BRAF mutational status. Analyses of tumor tissues confirmed that Spry1KO significantly reduced HIF1-α protein abundance. Interestingly, HIF-1α mRNA levels were minimally affected, thus suggesting that Spry1KO might impact on HIF-1α expression post-translationally. IP-MS identified about 50 Spry1 protein partners, and most of them were mitochondrial. Importantly, western blot analysis of cytosolic and mitochondrial proteins revealed that Spry1 was largely expressed in CM cell lines irrespective of BRAF mutation. Conclusions: Altogether, these data lead us to speculate that Spry1 might play a role in mitochondria homeostasis, thus impacting on hypoxia and angiogenesis in CM. Citation Format: Barbara Montico, Giorgio Giurato, Roberto Guerrieri, Annamaria Salvati, Francesca Colizzi, Lorena Baboci, Luca Sigalotti, Alessia Covre, Michele Maio, Agostino Steffan, Tuula A. Nyman, Alessandro Weisz, Maurizio Mongiat, Eva Andreuzzi, Elisabetta Fratta. Spry1 as a potential new hypoxia modulator in cutaneous melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4806.

Background and purpose Currently, human papillomavirus (HPV) positivity represents a strong prognostic factor for both reduced risk of relapse and improved survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). However, a subset of HPV-positive OPSCC patients still experience poor outcomes. Furthermore, HPV-negative OPSCC patients, who have an even higher risk of relapse, are still lacking suitable prognostic biomarkers for clinical outcome. Here, we evaluated the prognostic value of LINE-1 methylation level in OPSCC patients and further addressed the relationship between LINE-1 methylation status and p53 protein expression as well as genome-wide/gene-specific DNA methylation. Results In this study, DNA was extracted from 163 formalin-fixed paraffin-embedded tissue samples retrospectively collected from stage III-IVB OPSCC patients managed with curative intent with up-front treatment. Quantitative methylation-specific PCR revealed that LINE-1 hypomethylation was directly associated with poor prognosis (5-year overall survival—OS: 28.1% for LINE-1 methylation p TP53) gene is often mutated and overexpressed in HPV-negative OPSCC. Since p53 has been reported to repress LINE-1 promoter, we then analyzed the association between p53 protein expression and LINE-1 methylation levels. Following p53 immunohistochemistry, results indicated that among HPV16-negative patients with p53 ≥ 50%, LINE-1 methylation levels declined and remained stable at approximately 43%; any HPV16-positive patient reported p53 ≥ 50%. Finally, DNA methylation analysis demonstrated that genome-wide average methylation level at cytosine–phosphate–guanine sites was significantly lower in HPV16-negative OPSCC patients who relapsed within two years. The subsequent integrative analysis of gene expression and DNA methylation identified 20 up-regulated/hypomethylated genes in relapsed patients, and most of them contained LINE-1 elements in their promoter sequences. Conclusions Evaluation of the methylation level of LINE-1 may help in identifying the subset of OPSCC patients with bad prognosis regardless of their HPV status. Aberrant LINE-1 hypomethylation might occur along with TP53 mutations and lead to altered gene expression in OPSCC.