Neurotrophin Pathway Receptors NGFR and TrkA Control Perineural Invasion, Metastasis, and Pain in Oral Cancer.

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.

Brush swab as a noninvasive surrogate for tissue biopsies in epigenomic profiling of oral cancer.

BACKGROUND: Oral squamous cell carcinoma (OSCC) has poor survival rates. There is a pressing need to develop more precise risk assessment methods to tailor clinical treatment. Epigenome-wide association studies in OSCC have not produced a viable biomarker. These studies have relied on methylation array platforms, which are limited in their ability to profile the methylome. In this study, we use MethylCap-Seq (MC-Seq), a comprehensive methylation quantification technique, and brush swab samples, to develop a noninvasive, readily translatable approach to profile the methylome in OSCC patients. METHODS: Three OSCC patients underwent collection of cancer and contralateral normal tissue and brush swab biopsies, totaling 4 samples for each patient. Epigenome-wide DNA methylation quantification was performed using the SureSelectXT Methyl-Seq platform. DNA quality and methylation site resolution were compared between brush swab and tissue samples. Correlation and methylation value difference were determined for brush swabs vs. tissues for each respective patient and site (i.e., cancer or normal). Correlations were calculated between cancer and normal tissues and brush swab samples for each patient to determine the robustness of DNA methylation marks using brush swabs in clinical biomarker studies. RESULTS: There were no significant differences in DNA yield between tissue and brush swab samples. Mapping efficiency exceeded 90% across all samples, with no differences between tissue and brush swabs. The average number of CpG sites with at least 10x depth of coverage was 2,716,674 for brush swabs and 2,903,261 for tissues. Matched tissue and brush swabs had excellent correlation (r = 0.913 for cancer samples and r = 0.951 for normal samples). The methylation profile of the top 1000 CpGs was significantly different between cancer and normal samples (mean p-value = 0.00021) but not different between tissues and brush swabs (mean p-value = 0.11). CONCLUSIONS: Our results demonstrate that MC-Seq is an efficient platform for epigenome profiling in cancer biomarker studies, with broader methylome coverage than array-based platforms. Brush swab biopsy provides adequate DNA yield for MC-Seq, and taken together, our findings set the stage for development of a non-invasive methylome quantification technique for oral cancer with high translational potential.

Targeting the endothelin axis as a therapeutic strategy for oral cancer metastasis and pain.

Metastasis reduces survival in oral cancer patients and pain is their greatest complaint. We have shown previously that oral cancer metastasis and pain are controlled by the endothelin axis, which is a pathway comprised of the endothelin A and B receptors (ETAR and ETBR). In this study we focus on individual genes of the pathway, demonstrating that the endothelin axis genes are methylated and dysregulated in cancer tissue. Based on these findings in patients, we hypothesize that ETAR and ETBR play dichotomous roles in oral carcinogenesis and pain, such that ETAR activation and silenced ETBR expression result in increased carcinogenesis and pain. We test a treatment strategy that targets the dichotomous functions of the two receptors by inhibiting ETAR with macitentan, an ETAR antagonist approved for treatment of pulmonary hypertension, and re-expressing the ETBR gene with adenovirus transduction, and determine the treatment effect on cancer invasion (i.e., metastasis), proliferation and pain in vitro and in vivo. We demonstrate that combination treatment of macitentan and ETBR gene therapy inhibits invasion, but not proliferation, in cell culture and in a mouse model of tongue cancer. Furthermore, the treatment combination produces an antinociceptive effect through inhibition of endothelin-1 mediated neuronal activation, revealing the analgesic potential of macitentan. Our treatment approach targets a pathway shown to be dysregulated in oral cancer patients, using gene therapy and repurposing an available drug to effectively treat both oral cancer metastasis and pain in a preclinical model.

Anti-cancer and analgesic effects of resolvin D2 in oral squamous cell carcinoma.

Oral cancer is often painful and lethal. Oral cancer progression and pain may result from shared pathways that involve unresolved inflammation and elevated levels of pro-inflammatory cytokines. Resolvin D-series (RvDs) are endogenous lipid mediators derived from omega-3 fatty acids that exhibit pro-resolution and anti-inflammatory actions. These mediators have recently emerged as a novel class of therapeutics for diseases that involve inflammation; the specific roles of RvDs in oral cancer and associated pain are not defined. The present study investigated the potential of RvDs (RvD1 and RvD2) to treat oral cancer and alleviate oral cancer pain. We found down-regulated mRNA levels of GPR18 and GPR32 (which code for receptors RvD1 and RvD2) in oral cancer cells. Both RvD1 and RvD2 inhibited oral cancer proliferation in vitro. Using two validated mouse oral squamous cell carcinoma xenograft models, we found that RvD2, the more potent anti-inflammatory lipid mediator, significantly reduced tumor size. The mechanism of this action might involve suppression of IL-6, C-X-C motif chemokine 10 (CXCL10), and reduction of tumor necrosis. RvD2 generated short-lasting analgesia in xenograft cancer models, which coincided with decreased neutrophil infiltration and myeloperoxidase activity. Using a cancer supernatant model, we demonstrated that RvD2 reduced cancer-derived cytokines/chemokines (TNF-α, IL-6, CXCL10, and MCP-1), cancer mediator-induced CD11b+Ly6G- myeloid cells, and nociception. We infer from our results that manipulation of the endogenous pro-resolution pathway might provide a novel approach to improve oral cancer and cancer pain treatment.

OPRM1 Methylation Contributes to Opioid Tolerance in Cancer Patients.

Cancer patients in pain require high doses of opioids and quickly become opioid-tolerant. Previous studies have shown that chronic cancer pain as well as high-dose opioid use lead to mu-opioid receptor downregulation. In this study we explore downregulation of the mu-opioid receptor gene (OPRM1), as a mechanism for opioid tolerance in the setting of opioid use for cancer pain. We demonstrate in a cohort of 84 cancer patients that high-dose opioid use correlates with OPRM1 hypermethylation in peripheral leukocytes of these patients. We then reverse-translate our clinical findings by creating a mouse cancer pain model; we create opioid tolerance in the mouse cancer model to mimic opioid tolerance in the cancer patients. Using this model we determine the functional significance of OPRM1 methylation on cancer pain and opioid tolerance. We focus on 2 main cells within the cancer microenvironment: the cancer cell and the neuron. We show that targeted re-expression of mu-opioid receptor on cancer cells inhibits mechanical and thermal hypersensitivity, and prevents opioid tolerance, in the mouse model. The resultant analgesia and protection against opioid tolerance are likely due to preservation of mu-opioid receptor expression on the cancer-associated neurons. PERSPECTIVE: We demonstrate that epigenetic regulation of OPRM1 contributes to opioid tolerance in cancer patients, and that targeted gene therapy could treat cancer-induced nociception and opioid tolerance in a mouse cancer model.