pyrimidinones and Mouth-Neoplasms

Many of the characteristics ascribed to cancer-associated fibroblasts (CAFs) are shared by activated, autophagic and senescent fibroblasts. Whilst most oral squamous cell carcinomas (OSCCs) are genetically unstable (GU-OSCC), genetically stable variants (GS-OSCC) have been described and, notably, CAF activation (myofibroblast differentiation) and senescence are characteristics particularly associated with GU-OSCCs. However, it is not known whether autophagy is disrupted in these cells or whether autophagy regulates the development of the myofibroblast and senescent phenotypes. In this study, we show that senescent CAFs from GU-OSCCs contained more autophagosomes than normal human oral fibroblasts (NHOFs) and CAFs from GS-OSCCs possibly due to autophagic impairment. Further, we show that deregulation of autophagy in normal fibroblasts, either by inhibition with autophagy inhibitor, SAR405, or activation with TGF-β1, induced fibroblast activation and senescence: In response to TGF-β1, autophagy was induced prior to the development of the activated and senescent phenotypes. Lastly, we show that both SAR405- and TGF-β1-treated NHOFs enhance OSCC cell migration but only TGF-β1-treated cells increase OSCC invasion through Matrigel, indicating that TGF-β1 has additional effects that are independent of fibroblast activation/senescence. These results suggest a functional role for autophagy in the development of myofibroblast and CAF phenotypes.

Topics: Autophagy; Carcinoma, Squamous Cell; Cell Differentiation; Cell Movement; Cellular Senescence; Fibroblasts; Humans; Mouth Neoplasms; Myofibroblasts; Neoplasm Invasiveness; Pyridines; Pyrimidinones; Transforming Growth Factor beta1

Epidermal growth factor receptor (EGFR) is a major driver of head and neck cancer, a devastating malignancy with a major sub-site in the oral cavity manifesting as oral squamous cell carcinoma (OSCC). EGFR is a glycoprotein receptor tyrosine kinase (RTK) whose activity is upregulated in >80% OSCC. Current anti-EGFR therapy relies on the use of cetuximab, a monoclonal antibody against EGFR, although it has had only a limited response in patients. Here, we uncover a novel mechanism regulating EGFR activity, identifying a role of the nuclear branch of the Wnt/β-catenin signaling pathway, the β-catenin/CBP axis, in control of post-translational modification of N-glycans on the EGFR. Genomic and structural analyses reveal that β-catenin/CBP signaling represses fucosylation on the antennae of N-linked glycans on EGFR. By employing nUPLC-MS/MS, we determined that malignant human OSCC cells harbor EGFR with a paucity of N-glycan antennary fucosylation, while indolent cells display higher levels of fucosylation at sites N420 and N579. Additionally, treatment with either ICG-001 or E7386, which are both small molecule inhibitors of β-catenin/CBP signaling, leads to increased transcriptional expression of fucosyltransferases FUT2 and FUT3, with a concomitant increase in EGFR N-glycan antennary fucosylation. In order to discover which fucosylated glycan epitopes are involved in the observed effect, we performed in-depth characterization of multiply-fucosylated N-glycans via tandem mass spectrometry analysis of the EGFR tryptic glycopeptides. Data are available via ProteomeXchange with identifier PXD017060. We propose that β-catenin/CBP signaling promotes EGFR oncogenic activity in OSCC by inhibiting its N-glycan antennary fucosylation through transcriptional repression of FUT2 and FUT3.

Topics: Animals; beta Catenin; Binding Sites; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Squamous Cell; Cell Line, Tumor; CREB-Binding Protein; ErbB Receptors; Fucose; Fucosyltransferases; Galactoside 2-alpha-L-fucosyltransferase; Gene Expression Regulation, Neoplastic; Humans; Mice; Models, Molecular; Mouth Neoplasms; Neoplasm Metastasis; Polysaccharides; Protein Structure, Tertiary; Pyrimidinones; Small Molecule Libraries; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

Cell division cycle 7 (Cdc7) plays important roles in the regulation of the initiation of DNA replication throughout S phase. Whether inhibition of Cdc7 has a direct antitumour effect in oral squamous cell carcinoma (OSCC) remains unclear. In this study, XL413, a novel Cdc7 inhibitor, markedly inhibited the viability of OSCC cells but not that of non-tumour primary cells. There was a synergistic effect between XL413 and DNA-damaging agents (e.g. cisplatin and 5-fluorouracil) on OSCC in vitro and in vivo. Moreover, XL413 exhibited a notable antitumour effect on OSCC patients with high Cdc7 expression in mini patient-derived xenografts model. The proliferation was significantly inhibited in OSCC cells after Cdc7 silencing. Cdc7 knockdown significantly induced apoptosis in OSCC cell lines. Furthermore, we demonstrated that Cdc7 was overexpressed and transcriptionally regulated by E2F1 in OSCC by using chromatin immunoprecipitation and luciferase assays. Our results reveal that XL413 has an excellent antitumour effect in OSCC. Importantly, it does not inhibit the proliferation of non-tumour cells. These findings suggest that the overexpression of Cdc7 promotes progression in OSCC and that inhibition of Cdc7 is a very promising therapy for OSCC patients.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Line; Cell Proliferation; Cell Survival; E2F1 Transcription Factor; Female; Humans; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Mouth Neoplasms; Protein Serine-Threonine Kinases; Pyrimidinones

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy characterized by tumor heterogeneity, locoregional metastases, and resistance to existing treatments. Although a number of genomic and molecular alterations associated with HNSCC have been identified, they have had limited impact on the clinical management of this disease. To date, few targeted therapies are available for HNSCC, and only a small fraction of patients have benefited from these treatments. A frequent feature of HNSCC is the inappropriate activation of β-catenin that has been implicated in cell survival and in the maintenance and expansion of stem cell-like populations, thought to be the underlying cause of tumor recurrence and resistance to treatment. However, the therapeutic value of targeting β-catenin activity in HNSCC has not been explored.. We utilized a combination of computational and experimental profiling approaches to examine the effects of blocking the interaction between β-catenin and cAMP-responsive element binding (CREB)-binding protein (CBP) using the small molecule inhibitor ICG-001. We generated and annotated in vitro treatment gene expression signatures of HNSCC cells, derived from human oral squamous cell carcinomas (OSCCs), using microarrays. We validated the anti-tumorigenic activity of ICG-001 in vivo using SCC-derived tumor xenografts in murine models, as well as embryonic zebrafish-based screens of sorted stem cell-like subpopulations. Additionally, ICG-001-inhibition signatures were overlaid with RNA-sequencing data from The Cancer Genome Atlas (TCGA) for human OSCCs to evaluate its association with tumor progression and prognosis.. ICG-001 inhibited HNSCC cell proliferation and tumor growth in cellular and murine models, respectively, while promoting intercellular adhesion and loss of invasive phenotypes. Furthermore, ICG-001 preferentially targeted the ability of subpopulations of stem-like cells to establish metastatic tumors in zebrafish. Significantly, interrogation of the ICG-001 inhibition-associated gene expression signature in the TCGA OSCC human cohort indicated that the targeted β-catenin/CBP transcriptional activity tracked with tumor status, advanced tumor grade, and poor overall patient survival.. Collectively, our results identify β-catenin/CBP interaction as a novel target for anti-HNSCC therapy and provide evidence that derivatives of ICG-001 with enhanced inhibitory activity may serve as an effective strategy to interfere with aggressive features of HNSCC.

Topics: Animals; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Survival; Disease Progression; Epithelial Cells; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genomics; Head and Neck Neoplasms; Humans; Mice, Inbred C57BL; Mice, Nude; Molecular Targeted Therapy; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplastic Stem Cells; Peptide Fragments; Phenotype; Pyrimidinones; Sialoglycoproteins; Survival Analysis; Wnt Signaling Pathway; Zebrafish

Canine oral mucosal melanoma is an aggressive malignant neoplasm and is characterized by local infiltration and a high metastatic potential. The disease progression is similar to that of human oral melanomas. Whereas human cutaneous melanoma is primarily driven by activating mutations in Braf (60%) or Nras (20%), human mucosal melanoma harbors these mutations much less frequently. This makes therapeutic targeting and research modeling of the oral form potentially different from that of the cutaneous form in humans. Similarly, research has found only rare Nras mutations and no activating Braf mutations in canine oral melanomas, but they are still reliant on MAPK signaling. IQGAP1 is a signaling scaffold that regulates oncogenic ERK1/2 MAPK signaling in human Ras- and Raf- driven cancers, including melanomas. To investigate whether IQGAP1 is a potential target in canine melanoma, we examined the expression and localization of IQGAP1 in primary canine melanomas and canine oral melanoma cell lines obtained from the University of California-Davis. Using CRISPR/Cas9 knockout of IQGAP1, we examined effects on downstream ERK1/2 pathway activity and assayed proliferation of cell lines when treated with a peptide that blocks the interaction between IQGAP1 and ERK1/2. We observed that canine IQGAP1 is expressed and localizes to a similar extent in both human and canine melanoma by qPCR, Western blot, and immunofluorescence. Deletion of IQGAP1 reduces MAPK pathway activation in cell lines, similar to effects seen in human BrafV600E cell lines. Additionally, we demonstrated reduced proliferation when these cells are treated with a blocking peptide in vitro.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; CRISPR-Cas Systems; Dogs; Gene Knockout Techniques; Humans; Immunohistochemistry; MAP Kinase Signaling System; Melanoma; Mice; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mouth Neoplasms; Mutation; Oncogenes; Phosphorylation; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; ras GTPase-Activating Proteins

mTOR inhibition has emerged as a promising strategy for head and neck squamous cell carcinomas (HNSCC) treatment. However, most targeted therapies ultimately develop resistance due to the activation of adaptive survival signaling mechanisms limiting the activity of targeted agents. Thus, co-targeting key adaptive mechanisms may enable more effective cancer cell killing. Here, we performed a synthetic lethality screen using shRNA libraries to identify druggable candidates for combinatorial signal inhibition. We found that the ERK pathway was the most highly represented. Combination of rapamycin with trametinib, a MEK1/2 inhibitor, demonstrated strong synergism in HNSCC-derived cells in vitro and in vivo, including HNSCC cells expressing the HRAS and PIK3CA oncogenes. Interestingly, cleaved caspase-3 was potently induced by the combination therapy in PIK3CA+ cells in vitro and tumor xenografts. Moreover, ectopic expression of PIK3CA mutations into PIK3CA- HNSCC cells sensitized them to the pro-apoptotic activity of the combination therapy. These findings indicate that co-targeting the mTOR/ERK pathways may provide a suitable precision strategy for HNSCC treatment. Moreover, PIK3CA+ HNSCC are particularly prone to undergo apoptosis after mTOR and ERK inhibition, thereby providing a potential biomarker of predictive value for the selection of patients that may benefit from this combination therapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Drug Synergism; Female; Heterografts; Humans; MAP Kinase Signaling System; Mice; Mice, Nude; Molecular Targeted Therapy; Mouth Neoplasms; Pyridones; Pyrimidinones; RNA Interference; Sirolimus; TOR Serine-Threonine Kinases