The elucidation of the dysregulated molecular mechanisms driving HNSCC progression may afford a unique opportunity to identify novel targeted treatment options for HNSCC therapy and prevention. Several molecular targets have been tested in clinical trials and this chapter presents novel research on the efficacy of mTOR inhibition in cancer treatment and prevention. Other approaches that are emerging to targeting mTOR, for example with metformin also requires careful consideration.
Activation of the EGFR-PI3K-AKT-mTOR Signaling Network in Head and Neck Cancer
EGFR—presented in Chap. 6—regulates multiple intracellular signaling circuits, including the JAK/STAT, RAS/MAPK, and PI3K/AKT/mTOR pathways (Sharma et al. 2007; Tebbutt et al. 2013; Maulik et al. 2002). Among them, we have provided evidence that the persistent activation of PI3K/AKT/mTOR signaling circuitry is one of the most frequent dysregulated molecular events HNSCC lesions (Amornphimoltham et al. 2005; Molinolo et al. 2007a; Molinolo et al. 2012; Iglesias-Bartolome et al. 2013) (Fig. 7.1). This pathway involves the sequential activation of Phosphoinositide 3-kinase (PI3K), AKT, and mTOR. PI3Ks are grouped into three classes (I—III) according to their substrate preference and sequence homology (Cantley 2002). The class I PI3Ks are activated by growth factor tyrosine kinase receptors (class IA), such as EGFR, or by G protein coupled receptors (GPCRs) (class IB). Class IA PI3Ks are heterodimers of a p85 regulatory subunit and a p110 catalytic subunit. The direct product of PI3K activity is the lipid second messenger PtdIns (3,4,5)P3 (PIP3), which serves as docking sites for proteins that contain PH domains, including AKT and phosphoinositide-dependent kinase 1 (PDK1) (Cantley 2002; Sarbassov et al. 2006) which phosphorylates AKT in threonine 308 (pAKTT308) resulting in its activation. A second activation-specific AKT phosphorylation in serine 473 (pAKTS473) is targeted by mTOR as part of its complex 2 (mTORC2) (Sarbassov et al. 2006). In turn AKT plays a key role in the transmission of pro-proliferative and transforming pathways initiated by EGFR and multiple growth factor receptors, as well as by oncogenic active PI3K mutants (Luo et al. 2003).
- 7 Targeting the mTOR Signaling Circuitry in Head and Neck Cancer
Fig. 7.1 Genetics alterations in EGFR/PI3 K/mTOR signaling circuitry in HPV and HPV+ HNSCC lesion—precision therapeutic targets for HNSCC treatment
AKT prevents cell death by inactivating proapoptotic factors including BAD, procaspase-9 and Forkhead transcription factor family proteins (FOXOs), activates transcription factors that upregulate antiapoptotic genes, including NF-kB, inactivates p53 through Mdm2, and phosphorylates the cell cycle inhibitors p21CIP1/WAF1 and p27KIP1, thus increasing cell proliferation (Hennessy et al. 2005). AKT also phosphorylates and inhibits glycogen synthase kinase-3 (GSK3), thus enhancing p-catenin and cyclin D1 stabilization (Vivanco and Sawyers 2002). In this regard, we provided early evidence that AKT is persistently activated in the vast majority of HNSCC cases. Specifically, we showed that both experimental and human
HNSCCs and HNSCC-derived cell lines exhibit a remarkable increase in the levels of phosphorylated AKT (Amornphimoltham et al. 2004), and that blockade of PDK1, which acts upstream of AKT, inhibits tumor cell growth (Amornphimoltham et al. 2004; Patel et al. 2002). AKT activation was subsequently found to represent an early event in HNSCC progression. Active AKT can be detected in 50% of tongue preneoplastic lesions (Massarelli et al. 2005), and AKT activation can be considered an independent prognostic marker of poor clinical outcome in tongue and oropharyngeal HNSCC (Massarelli et al. 2005; Yu et al. 2007).
While AKT phosphorylates multiple downstream targets (see above), the emerging picture is that the ability of AKT to coordinate mitogenic and nutrient-sensing pathways controlling protein synthesis is a key mechanism by which AKT regulates cell proliferation. As depicted in Fig. 7.1, AKT phosphory- lates and inactivates the tumor-suppressor protein tuberous sclerosis complex protein 2 (TSC2), which forms a complex with tuberous sclerosis complex protein 1 (TSC1), and act together as a GTPase activating protein (GAP) for the small GTPase Rheb1 (Inoki et al. 2003, 2005a, b). AKT phosphorylation and inactivation of TSC2 results in increased levels of the GTP-bound (active) form of Rheb1, which in turn promotes the phosphorylation and activation of mTOR, also known as the mammalian target of rapamycin (Manning and Cantley 2003). Subsequently, mTOR phosphorylates key eukaryotic translation regulators, including p70-S6 kinase (p70S6K) and the eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) (Hay and Sonenberg 2004). The latter overrides the repressing activity of 4E-BP1 on the eukaryotic initiation factor 4E (eIF4E), resulting in enhanced translation of a subset of growth promoting genes (Hay and Sonenberg 2004). This is of particular relevance to HNSCC, as eIF4E gene amplification and protein overexpression is often associated with malignant progression (Sorrells et al. 1999), and eIF4E-positive surgical margins have increased risk of developing local recurrences (Nathan et al. 2002, 2004). Furthermore, supporting the relevance of the PI3K-AKT-mTOR pathway in HNSCC, we showed that the accumulation of the phosphorylated form of the ribosomal S6 protein, pS6, a typical downstream target of the mTOR pathway, is an early and one of the most frequent events in HNSCC (Molinolo et al. 2007b; Amornphimoltham et al. 2005). As part of an International HNSCC Tissue Array initiative, including HNSCC lesions from eight countries including Argentina, China, Japan, India, Mexico, South Africa, Thailand, and United States, we also observed that mTOR activation is a widespread event in HNSCC, irrespective of the risk factors associated with HNSCC initiation and progression in each country or geographical region (Molinolo Molinolo et al. 2007a, b). As described below, we also observed that inhibition of mTOR with specific inhibitors, such as rapamycin, provokes the rapid regression of multiple HNSCC xenografts (Amornphimoltham et al. 2005). These early findings provided a strong rationale for the preclinical and clinical evaluation of the efficacy of mTOR inhibiting strategies for HNSCC prevention and treatment.