The aims of the recently created Molecular Therapeutics Unit (MTU) is the identification of novel biochemical pathways altered in HNSCC leading to the discovery of small molecules which 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. Basic Aspects: Flavopiridol is a novel cdk inhibitor with antiproliferative activity in in vitro and in vivo models including HNSCC. Initial studies from our lab demonstrated that flavopiridol can induce cell cycle arrest, apoptosis, differentiation, modulate transcription and has anti-angiogenic properties. How exactly flavopiridol produces these effects is still unknown. Flavopiridol induces G1/S arrest by different mechanism(s), first, is an ATP competitor for most cdks tested (cdk1, cdk2, cdk4 and cdk7), second, by cdk dephosphorylation at threonine 160 (positive phosphorylation site required for cdk activity), and finally, by downregulation of cdk cofactors, cyclin D1 and cyclin D3. We have recently raised a polyclonal antiserum against the carboxyl terminus of cdk6 and demonstrated that flavopiridol inhibits cdk6 activity as efficiently as for other cdks. Moreover, to establish that cdk6 is inhibited in intact cells, we have raised 2 phospho-specific Rb polyclonal antiserum that detect Rb species phosphorylated at either the cdk2 site (Rb threonine 356) or the cdk4/6 site (Rb serine 780). Using the MCF10A cell line (only possesses cdk6 as the primary G1 kinase) we determined that flavopiridol is able to deplete Rb serine 780. If so, we will then determine the role of cdk6 inhibition in the G1/S arrest induced by flavopiridol. Flavopiridol can modulate transcriptional events (e.g. cyclin D1 and VEGF mRNA downregulation). In collaboration with David Price (University of Iowa) we demonstrated that flavopiridol inhibits P-TEFb (also known as cdk9/cyclin T) in vitro with a Ki of 3 nM. Interestingly, in contrast with all cdks tested so far, flavopiridol was not competitive with ATP in this reaction. P-TEFb is a required cellular cofactor for the human immunodeficiency virus (HIV-1) transactivator, Tat. As expected from P-TEFb inhibition, flavopiridol blocked Tat transactivation of the viral promoter in vitro. Furthermore, flavopiridol blocked HIV-1 replication in both single round and viral spread assays with an IC50 of less than 10 nM. Thus, this biochemical effect of flavopiridol should be tested in clinical trials of patients with HIV-related malignancies, including HIV lymphomas. Moreover, this novel biochemical effect of flavopiridol may explain the effects in gene expression such as cyclin D1, VEGF, etc. We are currently exploring the relationship between P-TEFb and regulation of gene expression by P-TEFb. In collaboration with F. Sarkar (Wayne State University), we investigated whether flavopiridol could be an effective agent against a series of isogenic breast- cancer cell lines having different levels of erbB-2 expression and differential invasion and metastatic characteristics. Flavopiridol was found to inhibit the growth of MDA-MB-435 (parental) and 435.eB (c-erbB-2 stable transfectants) cell lines. Induction of apoptosis was also in association with down-regulation of c-erbB-2. Gelatin zymography showed that flavopiridol inhibits the secretion of matrix metalloproteinase 2 and 9 in the breast cancer cells and that the inhibition of c-erbB-2 and MMPs may be responsible for the inhibition of cell invasion observed in flavopiridol-treated cells. Collectively, these molecular effects of flavopiridol, however, were found to be independent of c-erbB-2 overexpression, suggesting that flavopiridol may be effective even in erb2 positive breast cancer patients. From these results, we conclude that flavopiridol inhibits the growth and induces apoptosis in MDA-MB-435 breast cancer cells, and inhibits invasion and, thus, may inhibit metastasis of breast cancer cells. These findings suggest that flavopiridol may be an effective chemotherapeutic or preventive agent against breast cancer. Other studies in our unit demonstrated that flavopiridol induces apoptosis irrespective of functional p53 status and without modulation in the bcl-2/bax ratio. Ongoing studies demonstrate that flavopiridol activates several caspases. Moreover, pre-treatment with caspases inhibitors completely prevents apoptosis. To understand how these caspases are activated by flavopiridol, we are examining the role of the mitogen activated protein kinases pathways (MAPK), possible upstream regulators of caspases. Preliminary experiments demonstrated that in Jurkat cells treated with flavopiridol, MEK, p38 and JNK were activated just before the onset of apoptosis, suggesting that the enhanced activity of these kinases may activate caspases leading to apoptosis. Future studies will employ chemical inhibitors of MEK and p38 cascades. In order to challenge the system further, we are planning to transiently transfect Jurkat cells with constructs that encode different signaling pathways including several MAPK, AKT/PI3 kinase and BCL-2/Bax family members in order to discern the pathways that govern flavopiridol-induced activation of caspases. Translational/Clinical Aspects: We recently completed the first Phase I trial of bolus flavopiridol in cancer patients. Based on the encouraging results of this trial, we recently opened the accrual to the protocol """"""""Phase II trial of daily bolus flavopiridol for five consecutive days in patients with recurrent/metastatic squamous cell carcinoma of the Head and Neck (SCCHN)"""""""" in collaboration with Medicine Branch, NCI and NIDCD. UCN-01 is a novel protein kinase C (PKC) inhibitor with antitumor in vitro and in vivo models. UCN-01 has unique pharmacological features including induction of cell cycle arrest and apoptosis, and abrogation of G2 checkpoint imposed by DNA-damaging agents. Although some mechanistic questions are being answered, many important questions remain unknown. We recently completed the first Phase I trial of UCN-01 in cancer patients. Moreover, we reported that a patient that failed multiple chemotherapies including UCN-01, received a previously ineffective combination chemotherapy immediately after UCN-01 with total recovery within 2 weeks. However, the patient developed a fungemia and expired. At autopsy, there was no histological evidence of residual lymphoma. These results suggest that UCN-01 sensitized the lymphoma to the cytotoxic effects of EPOCH, possibly by modulating the """"""""threshold"""""""" for apoptosis, and may illustrate a new paradigm for reversal of drug resistance.
Showing the most recent 10 out of 29 publications
