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.

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.

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.

Ex vivo nonviral gene delivery of µ-opioid receptor to attenuate cancer-induced pain.

Virus-mediated gene delivery shows promise for the treatment of chronic pain. However, viral vectors have cytotoxicity. To avoid toxicities and limitations of virus-mediated gene delivery, we developed a novel nonviral hybrid vector: HIV-1 Tat peptide sequence modified with histidine and cysteine residues combined with a cationic lipid. The vector has high transfection efficiency with little cytotoxicity in cancer cell lines including HSC-3 (human tongue squamous cell carcinoma) and exhibits differential expression in HSC-3 (∼45-fold) relative to HGF-1 (human gingival fibroblasts) cells. We used the nonviral vector to transfect cancer with OPRM1, the µ-opioid receptor gene, as a novel method for treating cancer-induced pain. After HSC-3 cells were transfected with OPRM1, a cancer mouse model was created by inoculating the transfected HSC-3 cells into the hind paw or tongue of athymic mice to determine the analgesic potential of OPRM1 transfection. Mice with HSC-3 tumors expressing OPRM1 demonstrated significant antinociception compared with control mice. The effect was reversible with local naloxone administration. We quantified ß-endorphin secretion from HSC-3 cells and showed that HSC-3 cells transfected with OPRM1 secreted significantly more ß-endorphin than control HSC-3 cells. These findings indicate that nonviral delivery of the OPRM1 gene targeted to the cancer microenvironment has an analgesic effect in a preclinical cancer model, and nonviral gene delivery is a potential treatment for cancer pain.

TMPRSS2, a novel membrane-anchored mediator in cancer pain.

More than half of all cancer patients have significant pain during the course of their disease. The strategic localization of TMPRSS2, a membrane-bound serine protease, on the cancer cell surface may allow it to mediate signal transduction between the cancer cell and its extracellular environment. We show that TMPRSS2 expression is not only dramatically increased in the primary cancers of patients but TMPRSS2 immunopositivity is also directly correlated with cancer pain severity in these patients. TMPRSS2 induced proteolytic activity, activated trigeminal neurons, and produced marked mechanical hyperalgesia when administered into the hind paw of wild-type mice but not PAR2-deficient mice. Coculture of human cancer cells with murine trigeminal neurons demonstrated colocalization of TMPRSS2 with PAR2. These results point to a novel role for a cell membrane-anchored mediator in cancer pain, as well as pain in general.

Decitabine rescues cisplatin resistance in head and neck squamous cell carcinoma.

Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) reduces survival. In this study we hypothesized that methylation of key genes mediates cisplatin resistance. We determined whether a demethylating drug, decitabine, could augment the anti-proliferative and apoptotic effects of cisplatin on SCC-25/CP, a cisplatin-resistant tongue SCC cell line. We showed that decitabine treatment restored cisplatin sensitivity in SCC-25/CP and significantly reduced the cisplatin dose required to induce apoptosis. We then created a xenograft model with SCC-25/CP and determined that decitabine and cisplatin combination treatment resulted in significantly reduced tumor growth and mechanical allodynia compared to control. To establish a gene classifier we quantified methylation in cancer tissue of cisplatin-sensitive and cisplatin-resistant HNSCC patients. Cisplatin-sensitive and cisplatin-resistant patient tumors had distinct methylation profiles. When we quantified methylation and expression of genes in the classifier in HNSCC cells in vitro, we showed that decitabine treatment of cisplatin-resistant HNSCC cells reversed methylation and gene expression toward a cisplatin-sensitive profile. The study provides direct evidence that decitabine restores cisplatin sensitivity in in vitro and in vivo models of HNSCC. Combination treatment of cisplatin and decitabine significantly reduces HNSCC growth and HNSCC pain. Furthermore, gene methylation could be used as a biomarker of cisplatin-resistance.

Demethylating drugs as novel analgesics for cancer pain.

PURPOSE: In this study, we evaluated the analgesic potential of demethylating drugs on oral cancer pain. Although demethylating drugs could affect expression of many genes, we focused on the mu-opioid receptor (OPRM1) gene pathway, because of its role in pain processing. We determined the antinociceptive effect of OPRM1 re-expression in a mouse oral cancer model. EXPERIMENTAL DESIGN: Using a mouse oral cancer model, we determined whether demethylating drugs produced antinociception through re-expression of OPRM1. We then re-expressed OPRM1 with adenoviral transduction and determined if, and by what mechanism, OPRM1 re-expression produced antinociception. To determine the clinical significance of OPRM1 on cancer pain, we quantified OPRM1 methylation in painful cancer tissues and nonpainful contralateral normal tissues of patients with oral cancer, and nonpainful dysplastic tissues of patients with oral dysplasia. RESULTS: We demonstrated that OPRM1 was methylated in cancer tissue, but not normal tissue, of patients with oral cancer, and not in dysplastic tissues from patients with oral dysplasia. Treatment with demethylating drugs resulted in mechanical and thermal antinociception in the mouse cancer model. This behavioral change correlated with OPRM1 re-expression in the cancer and associated neurons. Similarly, adenoviral-mediated OPRM1 re-expression on cancer cells resulted in naloxone-reversible antinociception. OPRM1 re-expression on oral cancer cells in vitro increased ß-endorphin secretion from the cancer, and decreased activation of neurons that were treated with cancer supernatant. CONCLUSION: Our study establishes the regulatory role of methylation in cancer pain. OPRM1 re-expression in cancer cells produces antinociception through cancer-mediated endogenous opioid secretion. Demethylating drugs have an analgesic effect that involves OPRM1.

Novel animal models of acute and chronic cancer pain: a pivotal role for PAR2.

Targeted therapy to prevent the progression from acute to chronic pain in cancer patients remains elusive. We developed three novel cancer models in mice that together recapitulate the anatomical, temporal, and functional characteristics of acute and chronic head and neck cancer pain in humans. Using pharmacologic and genetic approaches in these novel cancer models, we identified the interaction between protease-activated receptor 2 (PAR2) and serine proteases to be of central importance. We show that serine proteases such as trypsin induce acute cancer pain in a PAR2-dependent manner. Chronic cancer pain is associated with elevated serine proteases in the cancer microenvironment and PAR2 upregulation in peripheral nerves. Serine protease inhibition greatly reduces the severity of persistent cancer pain in wild-type mice, but most strikingly, the development of chronic cancer pain is prevented in PAR2-deficient mice. Our results demonstrate a direct role for PAR2 in acute cancer pain and suggest that PAR2 upregulation may favor the development and maintenance of chronic cancer pain. Targeting the PAR2-serine protease interaction is a promising approach to the treatment of acute cancer pain and prevention of chronic cancer pain.

Stem cell markers as predictors of oral cancer invasion.

We previously showed that within primary tumors there exist subpopulations of cells expressing stem cell markers. Using immunofluorescence and western blotting, we examined the expression of stem cell markers tumor-rejection antigen 1-60 (TRA1-60) and octamer-binding transcription factor 3/ 4 (OCT3/4) to determine their relationship with cell invasiveness. Six human oral cancer cell lines were examined and a direct correlation was found between expression of these stem cell markers and invasion. Poor expression of E-cadherin and increased expression of N-cadherin was also found in TRA1-60- and OCT3/4- expressing cells. Phosphorylation of the major signaling molecule mitogen activated protein kinase (MAPK) was greatest in the TRA1-60- and OCT3/4- expressing cells. These results suggest that expression of specific stem cell markers in tumors may help guide a clinician's choice of treatment.

Analgesia targeting IB4-positive neurons in cancer-induced mechanical hypersensitivity.

UNLABELLED: Cancer patients often suffer from pain and most will be prescribed µ-opioids. µ-opioids are not satisfactory in treating cancer pain and are associated with multiple debilitating side effects. Recent studies show that µ and δ opioid receptors are separately expressed on IB4 (-) and IB4 (+) neurons, which control thermal and mechanical pain, respectively. In this study we investigated IB4 (+) and IB4 (-) neurons in mechanical and thermal hypersensitivity in an orthotopic mouse oral cancer model. We used a δ opioid receptor agonist and a P2X(3) antagonist to target IB4 (+) neurons and to demonstrate that this subset plays a key role in cancer-induced mechanical allodynia, but not in thermal hyperalgesia. Moreover, selective removal of IB4 (+) neurons using IB4-saporin impacts cancer-induced mechanical but not thermal hypersensitivity. Our results demonstrate that peripherally administered pharmacological agents targeting IB4 (+) neurons, such as a selective δ-opioid receptor agonist or P2X(3) antagonist, might be useful in treating oral cancer pain. PERSPECTIVE: To clarify the mechanisms of oral cancer pain, we examined the differential role of IB4 (+) and IB4 (-) neurons. Characterization of these 2 subsets of putative nociceptors is important for further development of effective clinical cancer pain relief.

Alphavbeta3 suppresses the RhoA-LIMK1 pathway in K1735 melanoma.

Mucocutaneous melanoma has a five-year survival rate of less than 10 percent. The alphavbeta3 integrin promotes invasion, which requires actin reorganization by cofilin. The authors previously showed that cofilin and alphavbeta3 promote invasion. K1735 melanoma has several clones, each with different levels of alphavbeta3. The authors found that expression of alphavbeta3 suppresses activation of RhoA thus inhibiting LIMK1 phosphorylation of cofilin. This indicates that alphavbeta3 integrin suppresses the RhoA/ ROCK/LIMK1 pathway.

Re-expression of the methylated EDNRB gene in oral squamous cell carcinoma attenuates cancer-induced pain.

Endothelin-1 is a vasoactive peptide that activates both the endothelin A (ET(A)) and endothelin B (ET(B)) receptors, and is secreted in high concentrations in many different cancer environments. Although ET(A) receptor activation has an established nociceptive effect in cancer models, the role of ET(B) receptors on cancer pain is controversial. EDNRB, the gene encoding the ET(B) receptor, has been shown to be hypermethylated and transcriptionally silenced in many different cancers. In this study we demonstrate that EDNRB is heavily methylated in human oral squamous cell carcinoma lesions, which are painful, but not methylated in human oral dysplasia lesions, which are typically not painful. ET(B) mRNA expression is reduced in the human oral squamous cell carcinoma lesions as a consequence of EDNRB hypermethylation. Using a mouse cancer pain model, we show that ET(B) receptor re-expression attenuates cancer-induced pain. These findings identify EDNRB methylation as a novel regulatory mechanism in cancer-induced pain and suggest that demethylation therapy targeted at the cancer microenvironment has the potential to thwart pain-producing mechanisms at the source, thus freeing patients of systemic analgesic toxicity.

Nerve growth factor links oral cancer progression, pain, and cachexia.

Cancers often cause excruciating pain and rapid weight loss, severely reducing quality of life in cancer patients. Cancer-induced pain and cachexia are often studied and treated independently, although both symptoms are strongly linked with chronic inflammation and sustained production of proinflammatory cytokines. Because nerve growth factor (NGF) plays a cardinal role in inflammation and pain, and because it interacts with multiple proinflammatory cytokines, we hypothesized that NGF acts as a key endogenous molecule involved in the orchestration of cancer-related inflammation. NGF might be a molecule common to the mechanisms responsible for clinically distinctive cancer symptoms such as pain and cachexia as well as cancer progression. Here we reported that NGF was highly elevated in human oral squamous cell carcinoma tumors and cell cultures. Using two validated mouse cancer models, we further showed that NGF blockade decreased tumor proliferation, nociception, and weight loss by orchestrating proinflammatory cytokines and leptin production. NGF blockade also decreased expression levels of nociceptive receptors TRPV1, TRPA1, and PAR-2. Together, these results identified NGF as a common link among proliferation, pain, and cachexia in oral cancer. Anti-NGF could be an important mechanism-based therapy for oral cancer and its related symptoms.

EMMPRIN expression in oral SCC is regulated by FYN kinase.

This study shows that the expression of the extracellular matrix metalloproteinase inducer (EMMPRIN) in oral squamous cell carcinoma cells (SCC) depends upon activation of the Src Family kinaseFyn; and that EMMPRIN and ß6 form a complex that requires active Fyn and the full length ß6 integrin cytoplasmic domain. Fyn is also important for matrix remodeling as it regulates both matrix type 1 metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-1 and -2 (TIMP1/2). The tumor promoter/suppressor caveolin-1, which associates with MT1-MMP, also requires FYN activation for expression. Lastly, EMMPRIN expression can act as a readout for the mitogen-activated protein kinase (MAPK) pathway, since when MAPK is blocked, so is the expression of EMMPRIN. In oral cancer, the activation of FYN occurs post ß6 integrin ligand binding. That the activation of FYN drives EMMPRIN expression and several important pathways associated with invasive oral SCC is now demonstrated.

Expression of Fyn kinase modulates EMT in oral cancer cells.

Oral squamous cell carcinoma (SCC) is an aggressive tumor with a poor 5-year survival rate. Oral SCC can undergo epithelial to mesenchymal transition (EMT). We previously showed that the epithelial integrin alphavbeta6 complexes with Fyn kinase in oral SCC to promote EMT. Using immunofluorescence microscopy and Western blotting, we evaluated whether the expression of specific markers of EMT were influenced by modulating serum concentration (ie. growth factors). The SCC cultures were grown under contrasting levels of serum. In low serum (1%), Fyn promoted EMT; whereas suppression of Fyn kinase promoted the epithelial phenotype. However, when the SCC cells were grown in 10% serum, activation of Fyn had the reverse effect. Lastly, cell migration was evaluated under low serum conditions (1% FBS). Activation of Fyn promoted SCC cell migration and its suppression thwarted SCC migration toward FN. These results indicate that the activation of Fyn kinase as well as local growth factor concentration modulate EMT in oral SCC.

Identification of {alpha}v{beta}6-positive stem cells in oral squamous cell carcinoma.

Oral squamous cell carcinoma (SCC) is composed of a heterogeneous population of cells which range anywhere from epithelial to mesenchymal in phenotype. Several oral cancer specimens with antibodies to TRA160, a marker of pluripotent cells, were screened. Compared with the well differentiated lesions, pluripotent cells were more numerous in specimens from poorly differentiated tumors. In vitro, the expression of TRA160 was much greater in invasive oral SCC9beta6 cells compared with the poorly invasive SCC9SN or SCC9beta6D1 cells, which express a truncated beta6. In vitro, pluripotent cells were instrumental in aggressively closing an experimental wound assay. Lastly, TRA-1-60+/beta6+ tumor cells which formed vascular-like structures in vivo were identified. SCC9beta6 cells formed interconnecting channels, whereas SCC9SN cells did not in an in vitro Matrigel angiogenesis assay. The results of this study clearly demonstrated the differential distribution of pluripotent stem cells in oral SCC and that the beta6 integrin may be an important regulatory component of the pluripotent phenotype.

The role of the integrin alpha v beta6 in regulating the epithelial to mesenchymal transition in oral cancer.

In this study, we evaluated whether the forced expression of beta6 integrin would modulate the epithelial to mesenchymal transition (EMT). When the full length beta6 integrin was expressed in poorly invasive squamous cell carcinoma SCC9 cells, the resulting SCC9/6 cells acquired a fibroblast-like morphology, increased expression of the mesenchymal marker vimentin and reduced expression of the epithelial markers keratin and E-cadherin. SCC9beta6D1 cells, which express a truncated form of beta6 subunit lacking the C-terminal 11 amino acids (AA), retained their epithelial morphology and did not alter vimentin or E-cadherin expression. This suggests that the full-length beta6 subunit can induce EMT in oral SCC cells. We previously showed that expression of beta6 increases both MMP-3 activation and tenascin-C expression and we now show that both molecules are MEK dependent. These results also demonstrate that the terminal 11 AA of beta6 contain information important for establishing an epithelial to mesenchymal transition.

Modulation of EGFR by oral squamous cell carcinoma cell lines.

Oral cancer is the sixth most frequent cancer worldwide. Prognosis for these patients remains poor. Recently, the epidermal growth factor receptor has been targeted as an adjunct to radiotherapy and surgery with limited success. The authors now present data suggesting that the alphanubeta6 integrin, which is a marker for aggressive oral cancer, may regulate epidermal growth factor receptor expression. The authors suggest perhaps targeting both alphanubeta6 and EGFR may provide additional benefits.

EMMPRIN modulates migration and deposition of TN-C in oral squamous carcinoma.

The extracellular matrix metalloproteinase inducer (EMMPRIN), found on the surface of many tumor cells, stimulates the production of matrix metalloproteinases (MMPs) by both fibroblasts and the tumor cells themselves. To evaluate its possible role as a tumor promoter, we first overexpressed EMMPRIN, by retroviral transduction, into poorly invasive squamous cell carcinoma (SCC) cells. Secondly, we knocked down its expression using small interfering RNA (siRNA) in invasive SCC cells. The cell lines were then re-evaluated for migration on fibronectin (FN). Overexpression of EMMPRIN, promoted motility, whereas the siRNA decreased migration. The MMP expression by these variant SCC cell lines was also manipulated by EMMPRIN. The expression of MMP-2, -3, and -9 coincided with the expression of EMMPRIN. Cocultures of SCC/peritumor fibroblasts (PTF) were used to investigate tenascin-C (TN-C) matrix deposition. The cocultures overexpressing EMMPRIN, deposited several fold greater levels of TN-C compared to the control cocultures. In addition, the siRNA cocultures deposited minimal amounts of TN-C. In the presence of the broad spectrum MMP inhibitor, GM6001, TN-C deposition by the EMMPRIN overexpressing cocultures was suppressed. Thus EMMPRIN regulates migration, MMP production by SCC cells and deposition of the TN-C matrix.

Alphavbeta3 integrin and cofilin modulate K1735 melanoma cell invasion.

Cytoskeletal reorganization is partially mediated through cofilin, an actin assembly regulatory protein. Cofilin activity is modulated by reversible phosphorylation at Ser3. In this study, using K1735 murine melanoma cells, we examined the relationship between beta3-integrin expression, phosphorylation of cofilin, and metalloproteinase production. The levels of phosphorylated cofilin were 10-fold higher in cells expressing alphavbeta3 than in alphavbeta3-negative cells when plated on vitronectin for 30 min. However, by 60 min, phosphorylation of cofilin was greater in the beta3-negative cells. Expression of the wild type (WT) or non-phosphorylatable cofilin (A3 mutant) increased melanoma cell migration on vitronectin and invasion through a reconstituted basement membrane. Expression of a pseudophosphorylated, poorly active cofilin (E3 mutant) reduced cell motility. Expression of active cofilin accelerated the phosphorylation of FAK at Y397 and at Y576, strongly implicating cofilin as a mediator of cell signaling. The expression of MT1-MMP and MMP2 was also increased by expression of wild type or A3 cofilin. A 50% reduction of both enzymes was observed by the expression of the E3 cofilin. Overexpression of non-phosphorylatable cofilin was sufficient to induce the expression of MT1-MMP and MMP2 in the beta3-negative M2Tbeta3 cells. Interestingly, the invasion of M2Tbeta3 cells could be sustained by overexpression of cofilin A3. These results suggest that the integrin alphavbeta3 and cofilin together regulate K1735 melanoma cell invasion.