Translational genomics and head and neck cancer: toward precision medicine.

Head and neck squamous cell carcinoma (HNSCC) comprise a wide spectrum of neoplasms with different tumor biologies, prognosis and response to therapies. Current tumor classification and traditional diagnostic methods (e.g. clinical assessment, histopathology) are limited in their capacity to determine prognosis and clinical decision-making. Despite recent improvements in treatment, the outcome for patients with HNSCC remains poor. Similar to most tumors, several patient-related factors, (e.g. genetics and environment) and disease-related factors (e.g. tumor location, TMN staging) play a significant role on survival. Thus, the problem in defining the prognosis is that the clinical course and response to treatment differ considerably among patients. Such interindividual variability is related to the heterogeneity of the tumor, genetic and epigenetic variations, thus reflecting the interaction of multiple biological components that result in a unique phenotype. Integrative genomics are developed to identify the molecular pathways leading to cancer at the individual level and find novel prognostic markers for HNSCC, hence tailoring a treatment accordingly. Such genetic-based personalized diagnosis allows tumor stratification and implementation of targeted therapy. Modern medicine includes new drugs that disrupt the implicated molecules and their signaling pathways. Here, we summarize the current state of knowledge that elucidates the translation of genetic data into clinical benefit.

Osteopontin posttranslational modifications, possibly phosphorylation, are required for in vitro bone resorption but not osteoclast adhesion.

Osteopontin (OPN), a phosphorylated bone matrix glycoprotein, is an Arg-Gly-Asp (RGD)-containing protein that interacts with integrins and promotes in vitro attachment of a number of cell types, including osteoclasts. Gene knockout experiments support the idea that OPN is important in osteoclastic activity. We hypothesize that posttranslational modifications (PTMs) of OPN can influence its physiological function. Previous studies have suggested that phosphorylation of OPN and bone sialoprotein (BSP) is necessary for promoting osteoclast adhesion. However, no reports have explored the importance of phosphoserines and other PTMs in OPN-promoted bone resorption. To study this question, we determined the activities of different forms of OPN and BSP in three in vitro assays: attachment of osteoclasts; formation of actin rings; and bone resorption. For each assay, cells were incubated for 4-24 h, in the presence or absence of RGDS or RGES peptides, to test the involvement of integrin binding. In addition to OPN, activities of milk OPN (fully phosphorylated) and recombinant OPN (rOPN, no phosphate) were compared. We purified two forms of OPN (OPN-2 and OPN-5), which differ in the level of phosphorylation, and compared their activities. For comparison, the activities of BSP and recombinant BSP (rBSP) were determined. All forms of OPN, including rOPN, significantly increased attachment of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts. BSP and rBSP also promoted cell attachment. After 4 h of incubation, the proportion of cells with actin rings was increased with OPN, milk OPN, and BSP. In the presence of RGDS peptide, osteoclast retraction and the disruption of actin rings were observed, whereas no effect was seen with RGES. In the resorption assay, the number of pits and the total resorbed area per slice were increased in the presence of OPN, milk OPN, and BSP. As in other assays, the OPN enhancement of resorption was inhibited by RGDS, but not RGES, peptides. Significantly, rOPN and rBSP did not promote bone resorption. OPN-5 promoted resorption to a greater extent than OPN-2, and milk OPN significantly stimulated resorption to a greater extent than OPN. Our data suggest that: (1) the RGD sequence of OPN is essential in OPN-mediated cell attachment, actin ring formation, and bone resorption; and (2) some form of PTM, possibly phosphorylation, is necessary for in vitro osteoclastic bone resorption, but not for cell attachment and actin ring formation.

Osteoclast adhesion and activity on synthetic hydroxyapatite, carbonated hydroxyapatite, and natural calcium carbonate: relationship to surface energies.

This study investigates the adhesion, cytoskeletal changes, and resorptive activity of disaggregated rat osteoclasts cultured on polished slices of three biomaterials: crystalline synthetic hydroxyapatite (HA), carbonated hydroxyapatite (C-HA), and natural calcium carbonate (C). The surface chemistry of each substrate was defined by X-ray diffraction and IR spectroscopy, surface wettability by the dispersive, and the polar components of the surface energies. Osteoclast adhesion was modulated by the polar component of the surface energy: fewer (p < 0.01) osteoclasts adhered to C-HA (97 +/- 20/slice, surface energy 9 +/- 5 mJ/m2) than to HA (234 +/- 16/slice, surface energy 44 +/- 2 mJ/m2) or to C (268 +/- 37/slice, surface energy 58 +/- 0.5 mJ/m2). Actin rings, which are the cytoskeletal structure essential for resorption, developed on all three materials. The area of the actin ring, which is resorbed by local acidification, and the osteoclast area, which reflects osteoclast spreading, were both greater in osteoclasts cultured on HA and C-HA than in those cultured on C. C was resorbed, but HA and C-HA were not. Thus, the surface energy plays an essential role in osteoclast adhesion, whereas osteoclast spreading may depend on the surface chemistry, especially on protein adsorption and/or on newly formed apatite layers. Resorption may be limited to the solubility of the biomaterial.

Rac-GTPase, osteoclast cytoskeleton and bone resorption.

The members of the Rho-GTPase subfamily, Rac1 and Rac2, are intimately involved in the organization of the cytoskeleton, and the p21-activated kinases or PAKs are targets of these proteins. Rac1 and Rac2 are also essential components of NADPH oxidase, the enzyme responsible for generating free radicals. The cytoskeleton modulates the adhesion of osteoclasts to bone and its subsequent resorption. These cells contain NADPH diaphorase activity, and free radicals influence bone resorption. The influence of Rac1, Rac2 and PAK1 on the cytoskeleton, resorbing activity and NADPH diaphorase activity of disaggregated rat osteoclasts was investigated by permeabilisation with saponin and introducing specific anti-Rac1, anti-Rac2 or anti-PAK1 antibodies. Rhodamine-phalloidin stain was used to identify actin in osteoclasts cultured on plastic slides, and the bone-slice method was used to measure resorption. Saponin permeabilisation did not affect the cytoskeletal organization or bone resorption. Anti-Rac antibodies caused dose- and time-dependent cytoskeletal changes. The osteoclasts rounded up and developed retraction fibers; actin rings were disrupted and large actin dots were seen at the periphery of the cells. Osteoclast resorptive activity was depressed after incubation with the antibodies. The total area resorbed by treated cells and the mean pit area were smaller than those of controls. Anti-PAK1 antibody caused similar changes. None of the antibodies altered the NADPH diaphorase activity. Thus, Rac-GTPases are present in rat osteoclasts and are involved in the organization of the actin cytoskeleton and in resorptive activity. These effects may be mediated by PAK1 kinase, but do not influence osteoclast NADPH diaphorase activity.