Genomic Database Analysis for Head and Neck Cancer Prevention Targets: MTOR Signal Transduction Pathway

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.

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Figure 1.

Mechanistic target of rapamycin (MTOR) pathway gene components, and their genetic changes in cancer. The + and − signs indicate typical physiological effects of each gene product on the pathway. Vertical arrows indicate change in gene expression in cancerous compared with normal tissue. IR: Insulin receptor; IRS: insulin receptor substrate 1; PIK3R1: phosphoinositide-3-kinase regulatory subunit 1; PIK3C: phosphatidylinositol 3-kinase subunits; PTEN: phosphatase and tensin homolog; AKT1: AKT serine/threonine kinase 1; TSC: tuberous sclerosis; PDK1: 3-phosphoinositide dependent protein kinase 1; RHEB: Ras homolog; MLST8: MTOR-associated protein; EIF4E: eukaryotic translation initiation factor 4E; EIF4EBP1: EIF4E binding protein 1; RPS6KB1: ribosomal protein S6 kinase B1.

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.