Activation of Akt at T308 and S473 in alcohol, tobacco and HPV-induced HNSCC: is there evidence to support a prognostic or diagnostic role?

HNSCC (Head and Neck Squamous Cell Carcinoma) includes cancers that involve the oral cavity, pharynx and larynx. Each year there are approximately 400 000 cases of cancer of the oral cavity and pharynx, with 160 000 cancers of the larynx, resulting in approximately 300 000 deaths [1]. It is the sixth most common type of cancer worldwide with a five year survival rate of 40-50%, which has shown only moderate improvement over the last two decades [2]. Two modifiable risk factors, tobacco use and alcohol consumption, are thought to explain approximately 75% of the incidence of HNSCC (up to 100 times higher for both) [3]; another important risk factor is HPV infection, which has been detected in around 20% of all cases [4].

Recent studies have focused on the genetic and epigenetic alterations of HNSCC, providing a better understanding of the molecular events underlying the pathogenesis of HNSCC [5, 6]. One of the most frequently altered signaling pathways in HNSCC is the PI3K/Akt cascade [6]. Akt, also known as PKB, is a serine-threonine protein kinase and is central to the phosphatidylinositol 3-kinase (PI3K) signaling pathway [7‐9]. PI3K is activated by tyrosine-kinase transmembrane receptors and other signaling intermediates, such as Ras oncogenes and G proteins [10]. PI3K then phosphorylates PtdIns (4,5) P2 (PIP2) yielding PtdIns (3,4,5) P3 (PIP3), which serves as an anchor for intracellular proteins (primarily mediated by pleckstrin homology domains), including Akt amongst others. Membrane-bound Akt is phosphorylated at T308 in the catalytic domain by the kinase PDK1 and at S473 in the regulatory domain by mTORC2 [11, 12]. Full Akt kinase activity is dependent upon phosphorylation at both T308 and S473 residues and this is greatly increased by growth factor receptor signaling [13]. PIP3 is converted back into PIP2 through the action of the lipid phosphatase PTEN, thus terminating the PI3K-initiated signal and avoiding further Akt activation [14]. Growth factor receptor over-expression [15, 16], mutation and down-regulation of PTEN protein [17] and amplification of the PIK3CA gene (the gene coding for the catalytic unit of PI3K) [18] can lead to increased Akt activity. Enhanced Akt activity has indeed been found in 20 to 60% of tumor samples and in the majority of HNSCC-derived cell lines [18‐21]. Once Akt is phosphorylated and activated, it is capable of phosphorylating multiple substrates generating diverse cellular processes, such as metabolism, proliferation, survival and protein synthesis [22].

An increasing number of mucosal changes and cellular atypia occur over large areas of the carcinogen-exposed upper aero-digestive tract epithelium, which initiate the stepwise carcinogenesis process in HNSCC. Acquisition of a transformed phenotype and accumulation of specific molecular genetic events are associated with this process [23‐28]; yet histopathological evaluation remains the time honored method in risk assessment of carcinoma lesions. In a search for better biological models of risk, Akt activation was recently identified as an early cellular response to carcinogen exposure and may be a significant step in environmental carcinogenesis [29]. Akt activation has also been found to correlate with squamous cell carcinoma progression from normal epithelium to invasive cancer [30].

These observations encouraged us to investigate the role of demographic, pathological and major risk factors (smoking, alcohol and HPV) of HNSCC patients on the activation of Akt (phosphorylation of Akt at Threonine 308 and Serine 473) and to determine their prognostic role.

To our knowledge this is the first report to show the relationships between the major risk factors for HNSCC (alcohol, smoking and HPV) and Akt activation (both at residues T308 and S473) at the protein level using an immunohistochemical staining method (IHC), in surgically resected specimens. Most studies to date have used IHC to assess the prognostic value of Akt activation in HNSCC, but have focused only on phosphorylation of residue S473. As the differential phosphorylation of Akt at the two sites may modulate downstream substrate selectivity and subsequent bioactivity [13], it is not surprising that Akt phosphorylated at any single site could perform certain cellular bioactivities. Two different mechanisms are involved in phosphorylating Akt, therefore overexpression or amplification of any components in these mechanisms may result in over-phosphorylation at any one of the two sites. It is therefore worth studying the phosphorylation status of Akt at both sites in HNSCC specimens to elucidate their different roles.

As only VEGFA positive HNSCC biopsy samples were selected in this study, no statistically significant correlation was found between VEGF and pAkt. The present study showed that Akt was significantly phosphorylated at T308 in VEGF positive HNSCC rather than S473. Alcohol and smoking were positively correlated with pAkt T308 activation but not with pAkt S473. Moreover, Akt activated at T308 showed a significant relationship with lymph node metastasis, which suggests that pAkt T308 may be concerned with invasion and metastasis. This data is similar to our in vitro data concerning the migration of tumor cells in response to VEGF, which suggests that migration of oral adeno-squamous cancer cells is dependent on Akt T308 phosphorylation. Our study also disclosed that Akt phosphorylated at both residues controls oral cancer cell motility [31], but it should be remembered that studies performed with cultured cells or tissue models may produce different results. In vivo, tumor progression requires both positive and reciprocal feedback between the components of the tissue microenvironment and cancer cells [32].

The activation of Akt in response to alcohol exposure is an important contributor to the molecular effects of excessive alcohol consumption [33]. In 2003, West showed that redundant Akt activation by nicotine and nicotine-derived nitrosamine ketone (NNK) could contribute to tobacco-related carcinogenesis [29]. A study by the Gonzalez group in 2005, revealed that Akt activation was correlated with concomitant PI3K accumulation and PTEN down-regulation in HNSCC, reflecting an early biochemical effect in response to nicotine [18]. Combined with these data, our study supports the basic hypothesis that Akt activation (especially at T308) is a key step in the progression of HNSCC caused by alcohol and smoking. HPV infection, another risk factor for HNSCC, was found to be negatively correlated with Akt activation at T308, as HPV positive HNSCC patients showed lower levels of pAkt T308. Non-drinking patients had lower levels of activated Akt at T308 too and there were more HPV positive patients among non-drinkers than amongst the heavy drinkers. Earlier epidemiologic research supports this data, that is non-smokers and light or non-drinkers are more likely to have tumors positive for HPV than are heavy smokers and drinkers [34]. Molinolo et al. [35] showed in their study that HPV positive HNSCC patients over-activate Akt at S473 and mTOR. Although we have not found any association between HPV infection and pAkt S473 activation, this may suggest that there are two different mechanisms of cancer progression initiated by alcohol, smoking and HPV. The Kelsey group study (2007), strongly supports the emerging view that the etiology of HPV related HNSCC is distinct from that of HNSCC tumors associated with smoking and drinking [36]. Increased Akt activation at T308 by excessive alcohol and smoking may be responsible for cancer development and progression, including metastasis, whereas HNSCC by HPV infection may over-activate Akt at S473 and be responsible for poor survival.

In this study we show that increased pAkt S473 levels in HNSCC are a strong predictor for poor patient outcome. In the multivariate Cox proportional hazard model, adjusted for well recognized prognostic indicators (e.g. tumor size and age), pAkt S473 status remained a strong predictor. This is corroborated by three other studies which have shown that pAkt activated at S473 is associated with poor prognosis in oral cancer [37‐39]. Although further molecular analysis is needed to investigate the mechanism of smoking and alcohol related HNSCC development, we can propose pAkt T308 as a reliable biomarker for smoking and alcohol induced HNSCC progression.

The predictive role of Akt activation in HNSCC suggests that targeting PI3K/Akt and mTORC2/Akt pathway along with RTK (receptor tyrosine kinase) might be a useful strategy for therapy in this disease. A large cohort with a longer follow-up of pre-neoplastic and HNSCC lesions is needed to more accurately define the role of Akt activation in carcinogenesis and to integrate this data into a risk model for carcinoma development and progression. In conclusion, our findings suggest that targeting Akt activation might be of interest as part of a combination therapy in HNSCC, as described earlier [40].

MI participated in the design of the study, carried out the immunohistochemistry and drafted the manuscript. MI performed the statistical analyses with and under direction of LC. MM was granted ethical approval for collection and use of the biopsies and directed the associated VEGF and HPV studies. IE and SJJ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. IE and SJJ were granted funding for the study and MI’s studentship. All authors have read and approved the final manuscript.