Abstract
Background: Type II diabetes agents have anticancer effects on head and neck squamous cell carcinoma (HNSCC). The mechanistic target of rapamycin (MTOR) pathway represents a putative target. Materials and Methods: We interrogated an Affymetrix HNSCC dataset for MTOR-related gene expression. Results: MTOR expression itself was unchanged, but various related genes demonstrated differential expression. Pathway promoters ras homolog (RHEB), MTOR-associated protein (MLST8), and ribosomal protein S6 kinase B1 (RPS6KB1) were up-regulated. Expression of growth suppressors tuberous sclerosis complex 2 (TSC2), programmed cell death 4 (PDCD4), and BCL2 apoptosis regulator-associated agonist of cell death (BAD) were reduced in HNSCC. Upstream, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), AKT serine/threonine kinase 1 (AKT1), and phosphatase and tensin homolog (PTEN) were up-regulated in cancer. Conclusion: Several MTOR pathway promoters and tumor suppressors were found to be differentially expressed, favoring MTOR pathway up-regulation in HNSCC. Genomic databases can be interrogated to identify intervention targets and endpoints in HNSCC trials.
Squamous cell carcinoma of the head and neck (HNSCC) is a devastating disease which affects approximately 53,000 patients in the United States each year, and results in over 10,000 deaths annually (1). A better molecular understanding of head and neck carcinogenesis would allow for the development of improved chemotherapy and chemoprevention agents. Our Molecular Oncology Program investigates carcinogenesis mechanisms to identify agents appropriate for intercepting the development of invasive cancer.
We have pursued head and neck cancer prevention by repurposing Food and Drug Administration-approved agents for type II diabetes, specifically pioglitazone and metformin, alone and in combination. Previous studies demonstrated at least a 35% reduction in both head and neck, and lung cancer incidence in diabetics treated with thiazolidinediones (2). Our group has demonstrated anticancer effects of these agents in preclinical studies as well as National Institutes of Health-sponsored head and neck chemoprevention trials. Metformin acts to antagonize mechanistic target of rapamycin (MTOR) pathway-associated head and neck carcinogenesis, and has demonstrated cancer prevention trends in patients with type II diabetes who are prescribed metformin (3). This effect may have utility in cancer chemoprevention.
MTOR is a cellular pathway involved in the interpretation of environmental cues and regulation of cell-cycle proliferation, growth, and metabolism, and may provide druggable targets for chemoprevention and cancer treatment in head and neck cancer. Previous work by Gutkind et al. uncovered up-regulation of the phosphoinositide 3-kinase (PI3-K), MTOR signaling pathway in HNSCC, and clinical trials of the MTOR inhibitor, rapamycin, yielded promising initial results (4). However, the emergence of acquired resistance to MTOR-specific targeting presents an opportunity to identify alternative MTOR pathway genes which may be viable pharmacotherapy targets.
Databases such as the Cancer Genome Atlas can be analyzed for the purpose of identifying treatment targets and disease biomarkers, based on mRNA expression in cancer specimens compared with controls. We examined the MTOR and associated signal transduction pathways in a cancer genomic database we developed using Affymetrix microarray data (5). We found we were indeed able to interrogate this database to identify expression levels of genes of interest in signal transduction pathways which appear important for chemoprevention. We conclude this type of analysis can be melded with other translational and basic science and epidemiology to have a better understanding of the pathophysiology of head and neck cancer for new agent development and treatment biomarkers.
Materials and Methods
Gene expression was compared between 41 HNSCC specimens and 13 mucosal controls using microarray analysis. Mucosal specimens were taken from healthy volunteers. Data were interrogated and analyzed from our previously published dataset (5), which contains all details of acquisition and analysis, human subject consent, experimental methods, and bioinformatic analytical techniques.
Expression of 19 genes of interest was analyzed in 41 HNSCC and 13 normal oral mucosal samples. MTOR and related pathway genes were identified in the original Affymetrix dataset and copied into a new Microsoft Excel spreadsheet (Microsoft Excel 2016; Microsoft Corporation, Redmond, WA, USA) to generate a specific database. A two-sided t-test was employed to compare HNSCC tumor gene expression to control gene expression for each expressed sequence tag (EST) of interest. Welch's correction was used, with a value of p<0.05 indicating significant difference. For genes with multiple ESTs, low-intensity ESTs were discarded if they were less than 20% expression of the maximum EST intensity for that gene. All statistical analysis was performed in Microsoft Excel 2016.
Results
To assess the gene-expression patterns of genes involved in the MTOR cellular signaling pathway, we re-interrogated a microarray dataset of 41 patients with HNSCC, compared with 13 mucosal controls obtained from the buccal mucosa of healthy individuals. The clinical characteristics of the 41 patients and 13 controls are displayed in Table I.
Mean expression levels of MTOR and related pathway genes of interest were compared between the tumor tissue from patients with HNSCC and buccal mucosa tissue from controls (6). Table II displays the change in expression of each gene assessed and the associated p-value. Gene-expression analysis of MTOR itself did not demonstrate differential expression in HNSCC specimens when compared with the controls (p=0.97). Pathway promoter ras homolog (RHEB) demonstrated up-regulation in the HNSCC samples in two ESTs (p<0.0001). MTOR associated protein (MLST8) also demonstrated up-regulation in cancer (p=0.045). The pathway suppressor tuberous sclerosis 1 (TSC1) did not demonstrate differential expression in HNSCC, however tuberous sclerosis 2 (TSC2) expression was reduced in the HNSCC samples (p=0.01), suggesting a potential mechanism for indirect up-regulation of MTOR activity.
MTOR targets eukaryotic translation initiation factor 4B (EIF4B) and EIF4B-binding protein (EIF4EBP1) were analyzed. Both EIF4B and EIF4BP demonstrated no change in expression (p>0.05). MTOR target eukaryotic translation initiation factor 4E (EIF4E) was down-regulated in one (p=0.04) of two ESTs. The ribosomal protein S6 kinases (RPS6KB1 and RPS6KB2) are also targets of mTOR. When analyzed, RPS6KB1 was up-regulated in one (p=0.037) of two ESTs and RPS6KB2 demonstrated down-regulation in HNSCC (p<0.0001). Analysis of one of the targets of RPS6KB, programmed cell death 4 (PDCD4), yielded down-regulation (p<0.0001) in HNSCC. PDCD4 targets MDM2 proto-oncogene (MDM2) and BCL2 apoptosis regulator-associated agonist of death (BAD) were also analyzed. HNSCC samples demonstrate no change in MDM2 expression (p>0.05) and reduced expression of BAD (p<0.01).
We also assessed upstream effectors of the MTOR pathway, specifically the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT pathway. Analysis of PI3K regulatory subunit 1 (PIK3R1) expression in HNSCC demonstrated up-regulation in two ESTs (p<0.044). Catalytic subunits PIK3CA (p=0.0012) and PIK3CD (p<0.0001) were up-regulated and PIK3CB was down-regulated (p=0.0018), however, in PIK3CD, one of the two queried ESTs had no change in expression (p>0.05). Downstream of PI3K, AKT serine/threonine kinase (AKT1) revealed up-regulation in the cancerous specimens (p=0.0021). PI3K pathway tumor suppressor phosphatase and tensin homolog (PTEN) was also up-regulated in HNSCC samples relative to the control in one (p=0.027) of two ESTs.
Discussion
MTOR is a serine/threonine kinase which functions as the catalytic subunit of MTORC1 and MTORC2, promoting cellular proliferation and growth (6). In this analysis, we found a number of significant gene-expression changes in pathway elements of MTOR associated with head and neck squamous tumors compared to buccal mucosa from healthy controls. MTOR itself is similarly expressed in tumor and normal mucosa, however, differential expression upstream and downstream of MTOR may result in increased cellular proliferation.
The gene products of RHEB and MLST8 act on MTOR to increase phosphotransferase activity. Gene products of TSC1 and TSC2 act together to convert RHEB into its inactive form (6), resulting in overall suppression of the MTOR pathway (Figure 1). RHEB and MLST8 up-regulation, as well as TSC2 down-regulation, are putative mechanisms to increase MTOR activity in head and neck carcinogenesis, despite no change in MTOR gene expression itself.
MTOR targets RPS6KB1 and 2 are involved the overall promotion of translation via the phosphorylation of ribosomal protein S6 (6). We discovered up-regulation of RPS6KB1 which may contribute to cellular proliferation in cancer. Increased S6 phosphorylation has been well-demonstrated in cancerous tissue (7), and our findings are in accordance with this. RPS6KB1 is also involved in cellular proliferation by its degradative effects on PDCD4 which promotes apoptosis when active (6). PDCD4 acts to suppress proliferation via MDM2 and BAD (8). In addition to the potential effects that up-regulation of RPS6KB1 would have on deactivating apoptosis via PDCD4 degradation, we found PDCD4 and BAD to be down-regulated in HNSCC, which would also contribute to increased cellular survival.
Upstream of MTOR, the PI3K/AKT pathway promotes cellular growth in the presence of environmental growth factors. PI3K is a heterodimer made up of PI3KR1 and PI3KC subunits. PI3K converts phosphatidylinositol (3,4)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-triphosphate (PIP3), recruiting and activating AKT (Figure 1) (6). The activation of the PI3K/AKT pathway ultimately increases MTORC1 activity (9). PTEN is a tumor suppressor which acts on the PI3K pathway by dephosphorylating PI3K (10). We observed up-regulation of PIK3R1, PIK3CA, PIK3CD, and PTEN in the cancer specimens. Up-regulation of PIK3R1 and PIK3C pathway genes may favor increased expression of PIP3, thus promoting the tumorigenic phenotype. Finally, we demonstrated up-regulation of AKT1 in cancerous specimens, a molecular finding that is well-demonstrated in studies of carcinogenesis (11), and specifically seen in head and neck cancer (4).
In summary, up-regulation of the MTOR complex with ultimate phosphorylation and activation of S6 can be promoted by alterations of expression of PIK3R1, TSC2, RHEB and RPS6KB1; the only inconsistent (but perhaps important) finding for pathway elements was the up-regulation of tumor suppressor PTEN.
The purpose of this project was to examine MTOR signal transduction, an important pathway in oral carcinogenesis progression (12). We have been investigating pioglitazone/metformin combination agent (Actoplus MET) (clinicaltrials.gov NCT02917629) in a window-of-opportunity trial and are collaborators for a recently completed metformin leukoplakia trial (clinicaltrials.gov NCT02581137). Some of the MTOR pathway elements we examined here are surrogate endpoints in these clinical trials. We identified differentially expressed genes that can be used for clinical trial endpoints, as well as potential therapeutic targets. In the future, it will be important to meld these genomic findings with other basic science technologies. One caveat of this initial study is that we used a low stringency for fold change. This way we captured the greatest number of targets for further analysis. In such a study, it would be quite useful to determine inositol triphosphate levels, which would give insight into the hyperfunctionality of MTOR. Ultimately, this analysis will help guide clinical trial biomarker modulation for current and future studies employing MTOR pathway inhibitors, such as rapamycin or metformin. Importantly, we feel the analysis of gene-expression databases with the goal of identifying pathways for therapy and prevention is feasible.
Acknowledgements
The Authors want to thank Beverly Wuertz, BA, for critical review of the manuscript. Grant support for the original project: American Cancer Society Institutional Research Grant IRG-58-00140IRG44.
Footnotes
Authors' Contributions
FO and CK designed the study. CK and FO participated in data analysis, and final article preparation. As the principal investigator, FO supervised the study. All Authors read and approved the final article.
Conflicts of Interest
The Authors declare they have no conflicts of interest regarding the study.
- Received June 15, 2020.
- Revision received July 8, 2020.
- Accepted July 10, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved