Abstract: Purpose: Epidemiologic studies have identified an increasing incidence of squamous cell carcinoma of the oral tongue (SCCOT) in younger patients. Experimental Design: DNA isolated from tongue tumors of young ( & lt;45 years, non-smokers) and old ( & gt;45 years) patients at was subjected to whole-exome sequencing and copy-number analysis. These data were compared with data from similar patients in the TCGA (The Cancer Genome Atlas) project. Results: In this study, we found that gene-specific mutation and copy-number alteration frequencies were similar between young and old patients with SCCOT in two independent cohorts. Likewise, the types of base changes observed in the young cohort were similar to those in the old cohort even though they differed in smoking history. TCGA data also demonstrate that the genomic effects of smoking are tumor site–specific, and we find that smoking has only a minor impact on the types of mutations observed in SCCOT. Conclusions: Overall, tumors from young patients with SCCOT appear genomically similar to those of older patients with SCCOT, and the cause for the increasing incidence of young SCCOT remains unknown. These data indicate that the functional impact of smoking on carcinogenesis in SCCOT is still poorly understood. Clin Cancer Res; 20(14); 3842–8. ©2014 AACR.

Abstract: As the guardian of genome, p53 plays important roles in maintaining the genome integrity, and TP53 mutations thereby often lead to genomic instability in cancers. In addition to the loss-of-function, many TP53 mutations can lead to the gain-of-functions (GOF) that often promote tumor development and tumor progression. As an effort to identify the mechanisms involved in mutant p53 GOF activities in head and neck squamous cell carcinoma (HNSCC), we performed an unbiased proteomic screen to identify the mutant p53 G245D interactome in HNSCC cells by immunoprecipitation, and quantitative proteomics using stable isotope labeling with amino acids in cell culture and liquid chromatography coupled with tandem mass spectrometry. Our results identified that MCM5, a member of replication licensing heterohexameric origin recognition complex minichromosome maintenance 2-7 (MCM2-7), interacts with mutant p53s, which suggests a possible role of mutant p53 in regulation of DNA replication. Consistent with this, we show that overexpression of mutant p53 in p53-depelted oral keratinocytes and HNSCC cells predisposes cells susceptibility to replication stress by inhibiting the dormant origin firing, leading to increased chromosomal instability (CIN) under replication stress. Moreover, by overexpressing and downregulating MCM5, we demonstrate that MCM5 modulates GOF mutant p53-mediated susceptibility to replication stress and CIN. Given the importance of MCM2-7 in replication licensing, initiation, and elongation, our discovery of the relationship between mutant p53 and MCM5 suggests that the MCM2-7 complex is one of the key downstream effectors, through which GOF mutant p53s regulate DNA replication and genomic instability in HNSCC. Citation Format: Mei Zhao, Tianxiao Wang, Chen Zhen, Jing Wang, Curtis R. Pickering, Junjie Chen, Jeffrey N. Myers, Ge Zhou. Gain-of-function mutant p53 predisposes head and neck keratinocytes and squamous cell carcinoma cells to replicative stress and genomic instability through minichromosome maintenance complex component 5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5289.

Abstract: TP53 mutations are highly prevalent in head and neck squamous cell carcinoma (HNSCC) and associated with increased resistance to conventional treatment primarily consisting of chemotherapy and radiation. Restoration of wild-type p53 function in TP53-mutant cancer cells represents an attractive therapeutic approach and has been explored in recent years. In this study, the efficacy of a putative p53 reactivator called COTI-2 was evaluated in HNSCC cell lines with different TP53 status. Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine in vitro and in vivo sensitivity of HNSCC cell lines with either wild-type, null, or mutant TP53 to COTI-2 alone, and in combination with cisplatin and/or radiation. Western blotting, cell cycle, live-cell imaging, RNA sequencing, reverse-phase protein array, chromatin immunoprecipitation, and apoptosis analyses were performed to dissect molecular mechanisms. Results: COTI-2 decreased clonogenic survival of HNSCC cells and potentiated response to cisplatin and/or radiation in vitro and in vivo irrespective of TP53 status. Mechanistically, COTI-2 normalized wild-type p53 target gene expression and restored DNA-binding properties to the p53-mutant protein in HNSCC. In addition, COTI-2 induced DNA damage and replication stress responses leading to apoptosis and/or senescence. Furthermore, COTI-2 lead to activation of AMPK and inhibition of the mTOR pathways in vitro in HNSCC cells. Conclusions: COTI-2 inhibits tumor growth in vitro and in vivo in HNSCC likely through p53-dependent and p53-independent mechanisms. Combination of COTI-2 with cisplatin or radiation may be highly relevant in treating patients with HNSCC harboring TP53 mutations.

Abstract: TP53 mutations are the most common cancer driver mutations among all cancers. Although some TP53 mutations lead to a loss of function of wild-type p53, many other TP53 mutations confer gain-of-function (GOF) activities, which promote cancer cell metastasis and pro-tumorigenic inflammation. Despite that many functional models of GOF mutant p53 (mutp53) have been proposed previously, the mechanisms involved in mutp53 GOF still remain largely elusive. Here we show that by directly targeting minichromosome maintenance complex component 5 (MCM5), a component of the hexametric DNA helicase MCM2-7 complex, GOF mutp53 predisposes cancer cells to replication stress and chromosomal instability, which leads to a tumor cell-autonomous and stimulator of interferon genes (STING)-dependent cytosolic DNA response that activates downstream non-canonical nuclear factor kappa light chain enhancer of activated B cell (NC-NF-κB) signaling. Furthermore, our results demonstrate that GOF mutp53-activated tumor cell-intrinsic STING-NC-NF-κB signaling not only stimulates tumor cell metastasis, but also promotes tumor immune resistance through fostering an immunosuppressive tumor microenvironment. Therefore, our findings that mutp53 exerts its GOF role through pro-tumorigenic MCM5-CIN-STING-NC-NF-κB signaling highlight the importance of TP53 and its inactivation in cancer genome evolution of genomic instability that drives tumor development and progression. Citation Format: Mei Zhao, Tianxiao Wang, Frederico O. Gleber-Netto, Zhen Chen, Daniel J. McGrail, Javier A. Gomez, Wutong Ju, Mayur A. Gadhikar, Wencai Ma, Li Shen, Ximing Tang, Sen Pathak, Maria G. Raso, Jared Burks, Shiaw-Yih Lin, Jing Wang, Asha S. Multani, Curtis R. Pickering, Junjie Chen, Jeffrey N. Myers, Ge Zhou. Mutant p53 gains oncogenic functions through a cytosolic DNA response [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 2585.

Abstract: Purpose: Development of extranodal extension (ENE) has been associated with poor survival in patients with oral cavity squamous cell carcinoma (OSCC). Here, we sought to confirm the role of ENE as a poor prognostic factor, and identify genomic and epigenetic markers of ENE in order to develop a predictive model and improve treatment selection. Experimental Design: An institutional cohort (The University of Texas MD Anderson Cancer Center) was utilized to confirm the impact of ENE on clinical outcomes and evaluate the genomic signature of primary and ENE containing tissue. OSCC data from The Cancer Genome Atlas (TCGA) were analyzed for the presence of molecular events associated with nodal and ENE status. Results: ENE was associated with decreased overall and disease-free survival. Mutation of the TP53 gene was the most common event in ENE+ OSCC. The frequency of TP53 mutation in ENE+ tumors was higher compared with ENE− tumors and wild-type (WT) TP53 was highly represented in pN0 tumors. pN+ENE+ patients had the highest proportion of high-risk TP53 mutations. Both primary tumors (PT) and lymph nodes with ENE (LN) exhibited a high rate of TP53 mutations (58.8% and 58.8%, respectively) with no significant change in allele frequency between the two tissue sites. Conclusions: ENE is one of the most significant markers of OSCC OS and DFS. There is a shift toward a more aggressive biological phenotype associated with high-risk mutations of the TP53 gene. Prospective clinical trials are required to determine whether TP53 mutational status can be used for personalized treatment decisions. Clin Cancer Res; 24(7); 1727–33. ©2018 AACR.

Abstract: The survival of patients with oral squamous cell carcinoma (OSCC) has not changed significantly in several decades, leading clinicians and investigators to search for promising molecular targets. To this end, we conducted comprehensive genomic analysis of gene expression, copy number, methylation, and point mutations in OSCC. Integrated analysis revealed more somatic events than previously reported, identifying four major driver pathways (mitogenic signaling, Notch, cell cycle, and TP53) and two additional key genes (FAT1, CASP8). The Notch pathway was defective in 66% of patients, and in follow-up studies of mechanism, functional NOTCH1 signaling inhibited proliferation of OSCC cell lines. Frequent mutation of caspase-8 (CASP8) defines a new molecular subtype of OSCC with few copy number changes. Although genomic alterations are dominated by loss of tumor suppressor genes, 80% of patients harbored at least one genomic alteration in a targetable gene, suggesting that novel approaches to treatment may be possible for this debilitating subset of head and neck cancers. Significance: This is the first integrated genomic analysis of OSCC. Only through integrated multiplatform analysis was it possible to identify four key pathways. We also discovered a new disease subtype associated with CASP8 and HRAS mutation. Finally, many candidate targetable events were found and provide hope for future genomically driven therapeutic strategies. Cancer Discov; 3(7); 770–81. ©2013 AACR. See related commentary by Iglesias-Bartolome et al., p. 722 This article is highlighted in the In This Issue feature, p. 705

Abstract: The solid tumor microenvironment includes nerve fibers that arise from the peripheral nervous system. Recent work indicates that newly formed adrenergic nerve fibers promote tumor growth, but the origin of these nerves and the mechanism of their inception are unknown. Here, by comparing the transcriptomes of cancer-associated trigeminal sensory neurons with those of endogenous neurons in multiple mouse models of oral cancer, we identified an adrenergic differentiation signature. We show that loss of TP53 leads to adrenergic transdifferentiation of tumor-associated sensory nerves through loss of the microRNA miR-34a. Tumor cells that expressed wild-type p53 protein released vesicles containing microRNAs that were taken up by neighboring neurons. MiR-34a blocks neuronal proliferation, and the neurons were maintained in their current state. By contrast, tumors that had a mutant versions of the gene encoding p53 released vesicles that lacked miR-34a. In this case, neurons increased in number in the vicinity of the tumor, and these cells were reprogrammed as adrenergic neurons that express the molecule noradrenaline. These neurons had more axonal branches than did those near tumors that expressed wild-type p53. Interactions between adrenergic neurons and the tumor aided cancer growth. Tumor growth was inhibited by sensory denervation or pharmacological blockade of adrenergic receptors, but not by chemical sympathectomy of pre-existing adrenergic nerves; and when mice received a transplant of p53-deficient tumor cells, treatment with a drug (carvedilol) that blocks adrenergic signaling pathways slowed tumor growth. A retrospective analysis of tumor samples from patients with oral cancer revealed that p53 status was associated with nerve density, which was in turn associated with poor clinical outcomes. Neural regulation represents an emerging targetable pathway for the treatment of cancer. The peripheral adrenergic nervous system has previously been shown to regulate cancer tumorigenesis. In contrast to previous findings using a prostate cancer mouse model, in our oral cancer mouse model, ablation of the sympathetic nervous system before tumor inoculation neither abrogated the development of adrenergic neo-nerves nor inhibited tumor growth. Our present study reveals that the emergence of adrenergic neonerves in the tumor microenvironment accompanies the initial phase of oral cavity squamous cell carcinoma (OCSCC) development in a transgenic OCSCC mouse models. We have identified crosstalk between the peripheral nervous system and head and neck tumors and described a phenotypic switch, induced by cancer cells, in which sensory nerves differentiate into adrenergic neo-neurons. This crosstalk between cancer cells and neurons represents mechanism by which tumor-associated neurons are reprogrammed towards an adrenergic phenotype that can stimulate tumor progression, and is a potential target for anticancer therapy. Our findings show that in p53-deficient tumors, an miRNA-based mechanism mediates neuronal responses to environmental cues and determines the fate of cancer-associated neurons. We have shown that axonal sprouting and autonomic reprogramming of existing nerves occur as a result of exosomal miRNA shuttling from cancer cells to neurons. These miRNAs orchestrate gene expression via combined dominantly negative (for example, miR-34a) and positive (for example, miR-21 and miR-324) effects, activating transcriptional programs that establish neuronal identity. In our mouse model of OCSCC, surgical ablation of sensory nerves prevented the development of these adrenergic neo-nerves; and as tumors evolve, neo-neural networks develop in and around the tumor stroma, providing signals that coordinate cancer progression. Our results thus show that the peripheral sensory nerves may be reprogrammed during the development of cancer in a manner similar to that of neural progenitors that initiate adrenergic neurogenesis during tumor formation. These results are consistent with recent preclinical data suggesting that sympathetic fibers accumulate in the normal vicinity of solid tumor tissues and infiltrate into the stroma. Furthermore, clinical data show that cancer patients treated with β-blockade have improved survival, supporting the role of adrenergic nerve activity in cancer progression. Although further studies will be required to dissect the molecular events that link tumor-associated neuritogenesis to cancer progression, our data raise the tantalizing possibility that drugs that target both axonal growth and the adrenergic nervous system could be useful for the treatment of cancer. Moreover, our discovery that the absence of functional p53 influences the formation of neighboring neurons might have relevance for interpreting reports showing that fluctuations in the levels of wild-type p53 are observed in nerve regeneration. Thus, these findings might have repercussions that reach beyond the field of cancer research to regenerative medicine. Citation Format: Moran Amit, Hideaki Takahashi, Mihnea-Paul Dragomir, Simone Anfossi, Antje Lindemann, Frederico Netto-Glebber, Samantha Tam, Abdullah Osman, Erik Kuntsen, Curtis R. Pickering, Mei Zhao, Xiayu Rao, Jing Wang, Deborah A. Silverman, Carlos Caulin, Assaf Zinger, Ennio Tasciotti, Patrick Dougherty, Adel El-Naggar, George A. Calin, Jeffrey N. Myers. Cancer takes a nerve: Loss of p53 drives neuron reprogramming in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG07.

Abstract: Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties in promoting cancer cell invasive growth and metastasis, but the mechanisms mediating these functions largely remain elusive. We show here that overexpression of the GOF mutant p53 G245D and other GOF p53 mutants enhances invasive cell growth of p53-deficient head and neck squamous cell carcinoma (HNSCC) UM-SCC-1 cells both in vitro 3D culture and in vivo orthotopic tumor mouse model. We demonstrate that the expression of the oncogenic forkhead transcription factor FOXM1 is upregulated by GOF mutant p53s in UM-SCC-1 cells and tumors. Moreover, we show that overexpression of GOF mutant p53 G245D decreases the inhibitory phosphorylation of FOXO3a, a tumor suppressive forkhead transcription factor, leading to its cytoplasmic accumulation. Consistent with the observation that FOXO3a negatively regulates FOXM1 expression, we demonstrate that overexpression FOXO3a decreases GOF mutant p53-mediated FOXM1 expression, whereas downregulation of FOXO3a partially restores decreased FOXM1 expression due to loss of GOF mutant p53. In support of the roles of FOXO3a-FOXM1 signaling axis in mutant p53's GOF signaling, we further show that overexpression of FOXO3a or downregulation of FOXM1 impairs GOF mutant p53-mediated cell invasion, whereas downregulation of FOXO3a, in part, rescues impaired cell invasion due to downregulated GOF mutant p53. Finally, given that AMP-activated protein kinase (AMPK) directly phosphorylates and promotes FOXO3a function, and that our prior demonstration that GOF mutant p53s inhibit AMPK, our current study establishes that GOF mutant p53-AMPK-FOX3a-FOXM1 signaling is one of important mechanisms through which mutant p53s gain oncogenic functions in HNSCCs. Citation Format: Noriaki Tanaka, Mei Zhao, Jiexin Zhang, Jing Wang, Ge Zhou, Jeffrey N. Myers. Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cell by targeting forkhead transcription factors FOXO3a and FOXM1 [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 73.

Abstract: Checkpoint kinase inhibitors (CHKi) exhibit striking single-agent activity in certain tumors, but the mechanisms accounting for hypersensitivity are poorly understood. We screened a panel of 49 established human head and neck squamous cell carcinoma (HNSCC) cell lines and report that nearly 20% are hypersensitive to CHKi monotherapy. Hypersensitive cells underwent early S-phase arrest at drug doses sufficient to inhibit greater than 90% of CHK1 activity. Reduced rate of DNA replication fork progression and chromosomal shattering were also observed, suggesting replication stress as a root causative factor in CHKi hypersensitivity. To explore genomic underpinnings of CHKi hypersensitivity, comparative genomic analysis was performed between hypersensitive cells and cells categorized as least sensitive because they showed drug IC50 value greater than the cell panel median and lacked early S-phase arrest. Novel association between CDKN2A/p16 copy number loss, CDK2 activation, replication stress, and hypersensitivity of HNSCC cells to CHKi monotherapy was found. Restoring p16 in cell lines harboring CDKN2A/p16 genomic deletions alleviated CDK2 activation and replication stress, attenuating CHKi hypersensitivity. Taken together, our results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from CHKi therapy. Significance: These results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from therapy with CHK inhibitors. Cancer Res; 78(3); 781–97. ©2017 AACR.