The aim of the Molecular Therapeutics Unit (MTU) is the identification of novel biochemical pathways altered in HNSCC leading to the discovery of small molecules that may modulate molecular events important for oral carcinogenesis, and thus may represent novel chemotherapeutic agents. Furthermore, the unit focuses in the preclinical assessment of biochemical and molecular parameters modulated by these small molecules, which may help to monitor the effects on tumor samples in patients with head and neck cancer receiving these novel treatment modalities. Finally, selected signaling agents developed in the unit are tested in early clinical trials in patients with advanced HNSCC and other malignancies for proof of principle testing, in order to assess whether the preclinical effects observed occur also in humans; novel effects observed in human trials are then, investigated in our unit, reverse translation. 1)Mechanism of antiproliferative effects of small molecules cell cycle modulators Perifosine (per) is a novel alkylphospholipid with antitumor properties, derived from miltefosine, an approved drug in Europe for the treatment of cutaneous malignancies. The exact mechanism of action is still unknown. We recently established that perifosine promotes cell cycle arrest by increase in the endogenous cdk inhibitor p21-waf1/cip1 protein levels. To discern the mechanism of p21 upregulation, northern blot studies revealed that the accumulation of p21 is transcriptional. The minimal p21 promoter region required for perifosine effects is identical to the region required for Ras to activate p21 promoter. This region contains several sp1 sites. Mutations in each specific Sp1 site abolished the activation by perifosine. Moreover, we demonstrated that perifosine, similar to Ras, activates the MEK/ERK pathway. Furthermore, perifosine is able to phosphorylate Sp1 in MAPK-dependent sites, and this phosphorylation is required for Sp1 increased DNA binding (as measured by gel shift analysis). Finally, the transcriptional induction of p21 by perifosine requires MEK/ERK activation as chemical or genetic inhibitors of MEK, (PD98059 or MEKAA, respectively) abolished p21 transactivation, p21 induction and increase Sp1 DNA binging activity, concluding that Per activates the p21 promoter via activation of MEK by modulation of Sp1. This effort was submitted recently and is being reviewed for publication. Efforts to develop CDK modulators led us to the discovery of a novel class of CDK inhibitors, the paullones. Initial studies demonstrated that paullones inhibit CDKs in vitro, thereby blocking cell-cycle progression. However, the exact mechanism for the antiproliferative effects of paullones was never explored. We demonstrated for the first time that the most potent paullone, alsterpaullone (Alp), induced apoptosis and promoted loss in clonogenicity in the Jurkat cell line. Alp caused early activation of both caspase-8 and -9, leading to cleavage of caspase-3 and PARP. Studies of mitochondrial membrane potential demonstrated the activation of caspase-9 by Alp follows mitochondrial perturbation. Thus, it appears that the main effect of Alp appears to be the intrinsic/mitochondrial pathway. In the presence of the general caspase inhibitor ZVAD, the cell-cycle effects of Alp are unaltered while apoptosis is blocked, suggesting that the CDK effects of Alp are not sufficient for Alp-induced apoptosis. Additional studies with paullones are warranted to further characterize their preclinical effects and to explore their potential use in the clinical setting. 2)Cell cycle derangements in human head and neck cancer: It became clear in the last decade that most human malignancies have an aberrant Rb pathway. Unfortunately, the exact mechanism responsible for this alteration is still unknown. Initial efforts to discern this aberration focused on cyclin D1. Cyclin D1 is a proto-oncogene that functions by inactivation of the retinoblastoma tumor suppressor protein. A common polymorphism in the cyclin D1 gene is associated with the production of an alternate transcript of cyclin D1, termed cyclin D1b. Both the polymorphism and the variant transcript are associated with increased risk for multiple cancers and the severity of a given cancer; however, the underlying activities of cyclin D1b have not been elucidated relative to the canonical cyclin D1a. In collaboration with Dr. Knudsen we demonstrated that, surprisingly, cyclin D1b protein does not inappropriately accumulate in cells and exhibits stability comparable to cyclin D1a. As expected, the cyclin D1b protein was constitutively localized in the nucleus, whereas cyclin D1a was exported to the cytoplasm in S-phase. Despite enhanced nuclear localization, cyclin D1b is a poor catalyst of RB phosphorylation/inactivation. However, cyclin D1b potently induced cellular transformation in contrast to cyclin D1a. In summary, we demonstrated that cyclin D1b specifically disrupts contact inhibition in a manner distinct from cyclin D1a. These data reveal novel roles for D-type cyclins in tumorigenesis. Based on this information we have raised a cyclin D1 specific antibody to screen head and neck tumors for the presence of this variant in order to determine the relevance of cyclin D1b in HNSCC.
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