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Oral cancer patients suffer pain at the site of the cancer. Calcitonin gene related polypeptide (CGRP), a neuropeptide expressed by a subset of primary afferent neurons, promotes oral cancer growth. CGRP also mediates trigeminal pain (migraine) and neurogenic inflammation. The contribution of CGRP to oral cancer pain is investigated in the present study. The findings demonstrate that CGRP-immunoreactive (-ir) neurons and neurites innervate orthotopic oral cancer xenograft tumors in mice. Cancer increases anterograde transport of CGRP in axons innervating the tumor, supporting neurogenic secretion as the source of CGRP in the oral cancer microenvironment. CGRP antagonism reverses oral cancer nociception in preclinical oral cancer pain models. Single-cell RNA-sequencing is used to identify cell types in the cancer microenvironment expressing the CGRP receptor components, receptor activity modifying protein 1 Ramp1 and calcitonin receptor like receptor (CLR, encoded by Calcrl). Ramp1 and Calcrl transcripts are detected in cells expressing marker genes for Schwann cells, endothelial cells, fibroblasts and immune cells. Ramp1 and Calcrl transcripts are more frequently detected in cells expressing fibroblast and immune cell markers. This work identifies CGRP as mediator of oral cancer pain and suggests the antagonism of CGRP to alleviate oral cancer pain. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) patients suffer from poor survival due to metastasis or locoregional recurrence, processes that are both facilitated by perineural invasion (PNI). OSCC has higher rates of PNI than other cancer subtypes, with PNI present in 80% of tumors. Despite the impact of PNI on oral cancer prognosis and pain, little is known about the genes that drive PNI, which in turn drive pain, invasion, and metastasis. In this study, clinical data, preclinical, and in vitro models are leveraged to elucidate the role of neurotrophins in OSCC metastasis, PNI, and pain. The expression data in OSCC patients with metastasis, PNI, or pain demonstrate dysregulation of neurotrophin genes. TrkA and nerve growth factor receptor (NGFR) are focused, two receptors that are activated by NGF, a neurotrophin expressed at high levels in OSCC. It is demonstrated that targeted knockdown of these two receptors inhibits proliferation and invasion in an in vitro and preclinical model of OSCC, and metastasis, PNI, and pain. It is further determined that TrkA knockdown alone inhibits thermal hyperalgesia, whereas NGFR knockdown alone inhibits mechanical allodynia. Collectively the results highlight the ability of OSCC to co‐opt different components of the neurotrophin pathway in metastasis, PNI, and pain. [ABSTRACT FROM AUTHOR]

Individual bacteria and shifts in the composition of the microbiome have been associated with human diseases including cancer. To investigate changes in the microbiome associated with oral cancers, we profiled cancers and anatomically matched contralateral normal tissue from the same patient by sequencing 16S rDNA hypervariable region amplicons. In cancer samples from both a discovery and a subsequent confirmation cohort, abundance of Firmicutes (especially Streptococcus) and Actinobacteria (especially Rothia) was significantly decreased relative to contralateral normal samples from the same patient. Significant decreases in abundance of these phyla were observed for pre-cancers, but not when comparing samples from contralateral sites (tongue and floor of mouth) from healthy individuals. Weighted UniFrac principal coordinates analysis based on 12 taxa separated most cancers from other samples with greatest separation of node positive cases. These studies begin to develop a framework for exploiting the oral microbiome for monitoring oral cancer development, progression and recurrence. [ABSTRACT FROM AUTHOR]

Background Metastasis to the cervical (neck) lymph nodes is one of the most significant clinical factors responsible for death from oral squamous cell carcinoma (SCC). Therefore, the lymph nodes are frequently removed when the tumor is excised (neck dissection), even though the majority of patients will not benefit from the extra surgery. Two subtypes of oral SCC distinguished by the presence of tumor genomic aberrations +3q, -8p, +8q and/or +20 differ in risk for metastasis - high for the 3q8pq20 subtype, harboring one or more of the aberrations and low for the non-3q8pq20 subtype, lacking these alterations. A prior analysis of the literature suggested genes differentially methylated in the two subtypes. Therefore, the goal of this study was to further investigate the methylation status of candidate biomarkers of the non- 3q8pq20 subtype, and evaluate their utility for identifying patients at low risk for metastasis. Methods Methylation status of genes in a cohort of 52 oral SCC patients with at least five year follow up was determined by pyrosequencing. Gene expression levels were determined by quantitative RT-PCR. Growth following re-expression of HOXA9 in cultured oral SCC cells was assessed by proliferation and colony formation assays. Results A pilot study evaluating methylation levels of HOXA9, MT1A and HOXA11 promoters in DNA from 12 tumors (six each of the 3q8pq20 and non-3q8pq20 subtypes) revealed that only HOXA9 was differentially methylated. Significant differences in methylation levels of HOXA9 were observed amongst the 52 oral SCCs with respect to genomic subtype and nodal status (p = 0.014, and p = 0.024, respectively, Wilcoxon rank sum test). High levels of HOXA9 methylation and low levels of expression in oral SCC cell lines were observed compared to HaCaT, a non-tumorigenic keratinocyte cell line. Re-expression of HOXA9 in the SCC4 oral cancer cell line resulted in diminished proliferation and colony formation. Conclusions HOXA9 methylation is frequent in oral cancers and levels are higher in tumors with greater risk of metastasis. Expression of HOXA9 is low in cells with high levels of methylation and reduced expression appears to confer a growth advantage. [ABSTRACT FROM AUTHOR]

Background: Metastasis to the cervical (neck) lymph nodes is one of the most significant clinical factors responsible for death from oral squamous cell carcinoma (SCC). Therefore, the lymph nodes are frequently removed when the tumor is excised (neck dissection), even though the majority of patients will not benefit from the extra surgery. Two subtypes of oral SCC distinguished by the presence of tumor genomic aberrations +3q, -8p, +8q and/or +20 differ in risk for metastasis - high for the 3q8pq20 subtype, harboring one or more of the aberrations and low for the non-3q8pq20 subtype, lacking these alterations. A prior analysis of the literature suggested genes differentially methylated in the two subtypes. Therefore, the goal of this study was to further investigate the methylation status of candidate biomarkers of the non-3q8pq20 subtype, and evaluate their utility for identifying patients at low risk for metastasis. Methods: Methylation status of genes in a cohort of 52 oral SCC patients with at least five year follow up was determined by pyrosequencing. Gene expression levels were determined by quantitative RT-PCR. Growth following re-expression of HOXA9 in cultured oral SCC cells was assessed by proliferation and colony formation assays. Results: A pilot study evaluating methylation levels of HOXA9, MT1A and HOXA11 promoters in DNA from 12 tumors (six each of the 3q8pq20 and non-3q8pq20 subtypes) revealed that only HOXA9 was differentially methylated. Significant differences in methylation levels of HOXA9 were observed amongst the 52 oral SCCs with respect to genomic subtype and nodal status (p = 0.014, and p = 0.024, respectively, Wilcoxon rank sum test). High levels of HOXA9 methylation and low levels of expression in oral SCC cell lines were observed compared to HaCaT, a non-tumorigenic keratinocyte cell line. Re-expression of HOXA9 in the SCC4 oral cancer cell line resulted in diminished proliferation and colony formation. Conclusions: HOXA9 methylation is frequent in oral cancers and levels are higher in tumors with greater risk of metastasis. Expression of HOXA9 is low in cells with high levels of methylation and reduced expression appears to confer a growth advantage. [ABSTRACT FROM AUTHOR]

Oral cancer patients experience pain at the site of the primary cancer. Patients with metastatic oral cancers report greater pain. Lack of pain identifies patients at low risk of metastasis with sensitivity = 0.94 and negative predictive value = 0.89. In the same cohort, sensitivity and negative predictive value of depth of invasion, currently the best predictor, were 0.95 and 0.92, respectively. Cancer pain is attributed to cancer-derived mediators that sensitize neurons and is associated with increased neuronal density. We hypothesized that pain mediators would be overexpressed in metastatic cancers from patients reporting high pain. We identified 40 genes overexpressed in metastatic cancers from patients reporting high pain (n = 5) compared to N0 cancers (n = 10) and normal tissue (n = 5). The genes are enriched for functions in extracellular matrix organization and angiogenesis. They have oncogenic and neuronal functions and are reported in exosomes. Hierarchical clustering according to expression of neurotrophic and axon guidance genes also separated cancers according to pain and nodal status. Depletion of exosomes from cancer cell line supernatant reduced nociceptive behavior in a paw withdrawal assay, supporting a role for exosomes in cancer pain. The identified genes and exosomes are potential therapeutic targets for stopping cancer and attenuating pain. [ABSTRACT FROM AUTHOR]