Cara B. Gonzales

Vanilloids induce oral cancer apoptosis independent of TRPV1

OBJECTIVE To investigate the mechanisms of vanilloid cytotoxicity and anti-tumor effects in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS Immunohistochemistry and qPCR analyses demonstrated expression of the TRP vanilloid type 1 (TRPV1) receptor in OSCC. Using cell proliferation assays, calcium imaging, and three mouse xenograft models, prototypical vanilloid agonist (capsaicin) and antagonist (capsazepine) were evaluated for cytotoxic and anti-tumor effects in OSCC. RESULTS OSCC cell lines treated with capsaicin displayed significantly reduced cell viability. Pre-treatment with capsazepine failed to reverse these effects. Moreover, capsazepine alone was significantly cytotoxic to tumor cells, suggesting the mechanism-of-action is independent of TRPV1 activation. This was further confirmed by calcium imaging indicating that TRPV1 channels are not functional in the cell lines tested. We then examined whether the observed vanilloid cytotoxicity was due to the generation of reactive oxygen species (ROS) and subsequent apoptosis. Induction of ROS was confirmed by flow cytometry and reversed by co-treatment with the antioxidant N-acetyl-cysteine (NAC). NAC also significantly reversed vanilloid cytotoxicity in cell proliferation assays. Dose-dependent induction of apoptosis with capsazepine treatment was demonstrated by FACS analyses and c-PARP expression in treated cells. Our in vivo xenograft studies showed that intra-tumoral injections of capsazepine exhibited high effectiveness in suppressing tumor growth with no identifiable toxicities. CONCLUSIONS These findings confirm TRPV1 channel expression in OSCC. However anti-tumor effects of vanilloids are independent of TRPV1 activation and are most likely due to ROS induction and subsequent apoptosis. Importantly, these studies demonstrate capsazepine is a potential therapeutic candidate for OSCC.

Epigenetic deregulation of the anaplastic lymphoma kinase gene modulates mesenchymal characteristics of oral squamous cell carcinomas

DNA hypermethylation of promoter CpG islands is associated with epigenetic silencing of tumor suppressor genes in oral squamous cell carcinomas (OSCCs). We used a methyl-CpG-binding domain protein capture method coupled with next-generation sequencing (MBDCap-seq) to survey global DNA methylation patterns in OSCCs with and without nodal metastasis and normal mucosa (total n = 58). Of 1462 differentially methylated CpG islands identified in OSCCs relative to normal controls, MBDCap-seq profiling uncovered 359 loci linked to lymph node metastasis. Interactive network analysis revealed a subset of these loci (n = 23), including the anaplastic lymphoma kinase (ALK) gene, are potential regulators and effectors of invasiveness and metastatic progression. Promoter methylation of ALK was preferentially observed in OSCCs without node metastasis, whereas relatively lower methylation levels were present in metastatic tumors, implicating an active state of ALK transcription in the latter group. The OSCC cell line, SCC4, displayed reduced ALK expression that corresponded to extensive promoter CpG island methylation. SCC4 treatment with demethylating agents induced ALK expression and increased invasion and migration characteristics. Inhibition of ALK activity in OSCC cells with high ALK expression (CAL27, HSC3 and SCC25), decreased cell growth and resulted in changes in invasive potential and mesenchymal marker expression that were cell-line dependent. Although ALK is susceptible to epigenetic silencing during oral tumorigenesis, overwriting this default state may be necessary for modulating invasive processes involved in nodal metastases. Given the complex response of OSCC cells to ALK inhibition, future studies are required to assess the feasibility of targeting ALK to treat invasive OSCCs.

Wittig derivatization of sesquiterpenoid polygodial leads to cytostatic agents with activity against drug resistant cancer cells and capable of pyrrolylation of primary amines

Many types of cancer, including glioma, melanoma, non-small cell lung cancer (NSCLC), among others, are resistant to proapoptotic stimuli and thus poorly responsive to current therapies based on the induction of apoptosis in cancer cells. The current investigation describes the synthesis and anticancer evaluation of unique C12-Wittig derivatives of polygodial, a sesquiterpenoid dialdehyde isolated from Persicaria hydropiper (L.) Delabre. These compounds were found to undergo an unprecedented pyrrole formation with primary amines in a chemical model system, a reaction that could be relevant in the biological environment and lead to the pyrrolation of lysine residues in the target proteins. The anticancer evaluation of these compounds revealed their promising activity against cancer cells displaying various forms of drug resistance, including resistance to proapoptotic agents. Mechanistic studies indicated that compared to the parent polygodial, which displays fixative general cytotoxic action against human cells, the C12-Wittig derivatives exerted their antiproliferative action mainly through cytostatic effects explaining their activity against apoptosis-resistant cancer cells. The possibility for an intriguing covalent modification of proteins through a novel pyrrole formation reaction, as well as useful activities against drug resistant cancer cells, make the described polygodial-derived chemical scaffold an interesting new chemotype warranting thorough investigation.

Synthetic and Biological Studies of Sesquiterpene Polygodial: Activity of 9‐Epipolygodial against Drug‐Resistant Cancer Cells

Polygodial, a terpenoid dialdehyde isolated from Polygonum hydropiper L., is a known agonist of the transient receptor potential vanilloid 1 (TRPV1). In this investigation a series of polygodial analogues were prepared and investigated for TRPV1‐agonist and anticancer activities. These experiments led to the identification of 9‐epipolygodial, which has antiproliferative potency significantly exceeding that of polygodial. 9‐Epipolygodial was found to maintain potency against apoptosis‐resistant cancer cells as well as those displaying the multidrug‐resistant (MDR) phenotype. In addition, the chemical feasibility for the previously proposed mechanism of action of polygodial, involving the formation of a Paal–Knorr pyrrole with a lysine residue on the target protein, was demonstrated by the synthesis of a stable polygodial pyrrole derivative. These studies reveal rich chemical and biological properties associated with polygodial and its direct derivatives. These compounds should inspire further work in this area aimed at the development of new pharmacological agents, or the exploration of novel mechanisms of covalent modification of biological molecules with natural products.

Competing Roles of TGFβ and Nma/BAMBI in Odontoblasts

Nma/BAMBI is a novel pseudoreceptor with homology to a TGFβ type I receptor that lacks a serine/threonine kinase domain. Nma/BAMBI functions as a dominant-negative protein that regulates reciprocal epithelial-mesenchymal interactions during organogenesis. Therefore, we hypothesized that Nma/BAMBI regulates TGFβ signaling and downstream gene expression during dentinogenesis. To test this hypothesis, we examined the downstream gene expression profiles of major dentin extracellular matrix proteins in response to Nma/BAMBI, and we examined the roles of Nma/BAMBI and TGFβ-1 during dentinogenesis. Overexpression of Nma/BAMBI in the mouse odontoblast-like cell line MD10-A2 down-regulated expression of DSPP by 66% and up-regulated expression of DMP1 four-fold. TGFβ treatment reversed Nma/BAMBI’s negative effect on DSPP expression. Furthermore, we demonstrated that TGFβ negatively regulates Nma/BAMBI’s expression levels in MD10-A2 odontoblast-like cells. Analysis of these data, together, indicates that TGFβ and Nma/BAMBI are inversely regulated and that the sequence of expression determines the net effect on downstream gene expression.

Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma

Squamous cell carcinoma (SCC) comprises 90% of all head and neck cancers and has a poor survival rate due to late-stage disease that is refractive to traditional therapies. Epidermal growth factor receptor (EGFR) is over-expressed in greater than 80% of head and neck SCC (HNSCC). However, EGFR targeted therapies yielded little to no efficacy in clinical trials. This study investigated the efficacy of co-targeting EGFR and the anaplastic lymphoma kinase (ALK) whose promoter is hypomethylated in late-stage oral SCC (OSCC). We observed increased ALK activity in late-stage human OSCC tumors and invasive OSCC cell lines. We also found that while ALK inhibition alone had little effect on proliferation, co-targeting ALK and EGFR significantly reduced OSCC cell proliferation in vitro. Further analysis showed significant efficacy of combined treatment in HSC3-derived xenografts resulting in a 30% decrease in tumor volumes by 14days (p<0.001). Western blot analysis showed that co-targeting ALK and EGFR significantly reduced EGFR phosphorylation (Y1148) in HSC3 cells but not Cal27 cells. ALK and EGFR downstream signaling interactions are also demonstrated by Western blot analysis in which lone EGFR and ALK inhibitors attenuated AKT activity whereas co-targeting ALK and EGFR completely abolished AKT activation. No effects were observed on ERK1/2 activation. STAT3 activity was significantly induced by lone ALK inhibition in HSC3 cells and to a lower extent in Cal27 cells. Together, these data illustrate that ALK inhibitors enhance anti-tumor activity of EGFR inhibitors in susceptible tumors that display increased ALK expression, most likely through abolition of AKT activation.

Hedgehog and TGFβ signaling converge on Gli2 to control bony invasion and bone destruction in oral squamous cell carcinoma

Oral Squamous Cell Carcinoma (OSCC) is the sixth most common cancer worldwide. OSCC invasion into the lymph nodes and mandible correlates with increased rates of recurrence and lower overall survival. Tumors that infiltrate mandibular bone proliferate rapidly and induce bone destruction. While survival rates have increased 12% over the last 20 years, this improvement is attributed to general advances in prevention, earlier detection, and updated treatments. Additionally, despite decades of research, the molecular mechanisms of OSCC invasion into the mandible are not well understood. Parathyroid Hormone-related Protein (PTHrP), has been shown to be essential for mandibular invasion in OSCC animal models, and our previous studies demonstrate that the transcription factor Gli2 increases PTHrP expression in tumor metastasis to bone. In OSCC, we investigated regulators of Gli2, including Hedgehog, TGFβ, and Wnt signaling to elucidate how PTHrP expression is controlled. Here we show that canonical Hedgehog and TGFβ signaling cooperate to increase PTHrP expression and mandibular invasion in a Gli2-dependent manner. Additionally, in an orthotopic model of mandibular invasion, inhibition of Gli2 using shRNA resulted in a significant decrease of both PTHrP expression and bony invasion. Collectively, our findings demonstrate that multiple signaling pathways converge on Gli2 to mediate PTHrP expression and bony invasion, highlighting Gli2 as a therapeutic target to prevent bony invasion in OSCC.

Thymol inhibits oral squamous cell carcinoma growth via mitochondria-mediated apoptosis

BACKGROUND Thymol is a transient receptor potential ankyrin subtype 1 channel, (TRPA1) agonist found in thyme and oregano. Thymol has antioxidant, anti-inflammatory, and antimicrobial properties; thus, thymol is added to many commercially available products including Listerine mouthwash. Thymol is also cytotoxic to HL-60 (acute promyelocytic leukemia) cells in vitro. Therefore, we evaluated the effects of thymol against oral squamous cell carcinoma (OSCC) and its anticancer mechanism-of-action. METHODS The antiproliferative effects of thymol in OSCC Cal27 cells were determined by MTS assays. Antitumor effects were evaluated in Cal27- and HeLa-derived mouse xenografts. Calcium imaging, mitochondrial transmembrane potential (ΔΨm) studies, and Western blot analysis of cleaved PARP (c-PARP) evaluated thymols mechanism-of-action. RESULTS Thymol had significant, long-lasting antiproliferative effects in vitro. In vivo, thymol displayed significant antitumor effects in Cal27-derived tumors. Thymols anticancer effects were confirmed in HeLa-derived xenografts demonstrating that thymol effects are not tumor-type specific. Calcium imaging verified calcium influx in Cal27 cells that were reversed with the TRPA1 antagonist, HC030031. However, no calcium influx was seen in HeLa cells indicating that TRP channels do not regulate thymol cytotoxicity. This was confirmed using cell viability assays in which pre-treatment with HC030031 had no effect on thymol cytotoxicity. Instead, ΔΨm studies revealed that thymol induces significant ΔΨm depolarization and apoptosis. CONCLUSION Our findings provide the first evidence of thymols novel antitumor effects against OSCC in vivo, which do not rely on TRPA1 activity. Instead, we show that thymol induces mitochondrial dysfunction and apoptosis and may be efficacious against multiple cancers.

Novel polygodial analogs P3 and P27: Efficacious therapeutic agents disrupting mitochondrial function in oral squamous cell carcinoma

Polygodial, a drimane sesquiterpenoid dialdehyde isolated as a pungent component of the water pepper Persicaria hydropiper, exhibits antifeedant, antimicrobial, anti-inflammatory and anticancer effects. Polygodial also activates transient receptor potential vanilloid subtype 1 (TRPV1) channels. Previously, we described the synthesis of a C12-Wittig derivative of polygodial, termed P3, with significant antiproliferative effects against multiple cancer types including oral squamous cell carcinoma (OSCC). In the present study, a more potent derivative, P27, with superior anti-proliferative effects in vitro and antitumor effects in Cal-27 derived xenografts is described. Polygodial, P3, and P27 all significantly decreased OSCC tumor growth, with P27 being equipotent with polygodial and P3 being the least efficacious. However, neither analog elicited the adverse effect observed with polygodial: Profound transient inflammation. Although P3 and P27 pharmacophores are based on polygodial, novel effects on OSCC cell cycle distribution were identified and shared anticancer effects that are independent of TRPV1 activity were observed. Polygodial elicits an S-phase block, whereas P3 and P27 lead to G2/M phase arrest. Pretreatment of OSCC cells with the TRPV1 antagonist capsazepine does not affect the antiproliferative activity of P3 or P27, indicating that TRPV1 interactions do not regulate OSCC cell proliferation. Indeed, calcium imaging studies identified that the analogs neither activate nor antagonize TRPV1. Behavioral studies using a rat model for orofacial pain confirmed that these analogs fail to induce nocifensive responses, indicating that they are non-noxious in vivo. All compounds induced a significant concentration-dependent decrease in the mitochondrial transmembrane potential and corresponding apoptosis. Considering that P27 is equipotent to polygodial with no TRPV1-associated adverse effects, P27 may serve as an efficacious novel therapy for OSCC.

Abstract 3582: Epidermal growth factor receptor and anaplastic lymphoma kinase bypass signaling in oral squamous cell carcinoma in vivo

Oral squamous cell carcinoma (OSCC) is an insidious disease with poor prognosis. This is due to invasive disease that is refractive to conventional therapies. We reported a gene-set of hypomethylated loci in metastatic OSCC involved in Epidermal Growth Factor Receptor (EGFR) signaling. EGFR over-expression is an early event in OSCC however therapies targeting EGFR have had dismal results. Our studies showed hypomethylation of Anaplastic Lymphoma Kinase (ALK) correlates with metastatic OSCC. ALK and EGFR signaling drive glioblastoma and non-small cell lung cancers (NSCLC). Furthermore, NSCLC that develop resistance to ALK inhibition demonstrate activation of EGFR bypass signaling. Based upon these findings, we hypothesize that simultaneous targeting of both ALK and EGFR in OSCC will yield greater anti-tumor activity than single drug therapies. Our objective was to assess expression and activation of EGFR and ALK signaling pathways in response to therapies targeting EGFR and/or ALK in vitro and in mouse OSCC xenograft models. Expression of total and activated EGFR and ALK was analyzed in HSC3 cells (invasive) and SCC4 cells (non-invasive) using western blot analysis. Effects on downstream signaling molecules (AKT, STAT3, and RAS) were also evaluated following treatments with TAE684 (ALK inhibitor) and/or Gefitinib (EGFR inhibitor). Anti-tumor effects of TAE684 and/or Gefitinib were evaluated in mouse OSCC xenograft models using HSC3 and SCC4 cell lines. This study revealed that HSC3 and SCC4 cell lines have unique growth, invasion, and signaling profiles resulting in varying responses to treatments targeting ALK and/or EGFR. HSC3 cells express ALK and have active EGFR whereas SCC4 cells have low ALK expression and low EGFR activity. Subsequently, HSC3 xenografts were sensitive to Gefitinib and co-targeting ALK resulted in an additive effect. Targeting ALK alone had no effect on tumor growth. Similar to NSCLC, western blot analysis revealed that ALK inhibition alone induces increased activation of EGFR bypass signaling in both HSC3 and SCC4 cells. In contrast SCC4 xenografts failed to respond to TAE684 and/or Gefitinib treatments. Co-treatment with TAE684 and Gefitinib down regulated the already low EGFR activity. Downstream effector molecules AKT and STAT3 had reduced activity in response to co-treatments in both HSC3 and SCC4 cell lines. Conversely, MAPK activity increased with all treatments in both cell lines. Therefore, we conclude that OSCC with active EGFR and ALK signaling are responsive to combination treatments targeting ALK and EGFR resulting in more efficacious anti-tumor effects than single drug therapies. Furthermore, single anti-ALK therapy induces compensatory EGFR activation and has no effect on tumor growth. Additional parallel pathways may account for elevated MAPK activity in response to all treatments and warrants further investigation. Citation Format: Cara B. Gonzales, Heping Chen, Jorge J. De La Chapa, Nameer B. Kirma. Epidermal growth factor receptor and anaplastic lymphoma kinase bypass signaling in oral squamous cell carcinoma in vivo . [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3582. doi:10.1158/1538-7445.AM2015-3582