p16 Protein and gigaxonin are associated with the ubiquitination of NFκB in cisplatin-induced senescence of cancer cells.

The molecular mechanism of p16-mediated senescence in cisplatin-treated cancer cells is not fully understood. Here we show that cisplatin treatment of head and neck cancer cells results in nuclear transport of p16 leading to a molecular modification of NFκB. Chromatin immunoprecipitation assays show that this modification is associated with the inhibition of NFκB interacting with its DNA binding sequences, leading to decreased expression of NFκB-transcribed proteins. LCMS proteomic analysis of LAP-TAP-purified proteins from HeLa cells containing a tetracycline-inducible GFP-S peptide-NFκB expression system identified gigaxonin, an ubiquitin E3 ligase adaptor, as an NFκB-interacting protein. Immunoblotting and siRNA studies confirmed the NFκB-gigaxonin interaction and the dependence of this binding on p16-NFκB binding. Using gel shift assays, we have confirmed p16-NFκB and gigaxonin-NFκB interactions. Furthermore, we have observed increased NFκB ubiquitination with cisplatin treatment that is abolished in the absence of p16 and gigaxonin expression. Analysis of 103 primary tumors has shown that increased nuclear p16 expression correlates with enhanced survival of head and neck cancer patients (p < 0.0000542), indicating the importance of nuclear p16 expression in prognosis. Finally, p16 expression is associated with reduced cytokine expression and the presence of human papilloma virus in chemoradiation-sensitive basaloid tumors. However, the absence of p16 expression is associated with enhanced cytokine expression and the absence of human papilloma virus in aggressive tumors. These results clearly demonstrate that nuclear p16 and gigaxonin play an important role in chemosensitivity of head and neck cancers through ubiquitination of NFκB.

Regulation of prostate-specific antigen expression by the junctional adhesion molecule A.

OBJECTIVE: Prostate-specific antigen (PSA) is a protein specifically expressed in prostate cells. Therefore, the expression levels of PSA in the blood are an important indicator when diagnosing prostate cancer. Defining the mechanism of PSA expression in prostate cells will be helpful for interpreting the expression of this protein during prostate cancer progression. Reports show that a membrane protein, claudin-7 (CLDN-7), is involved in the expression of PSA. However, the mechanism by which CLDN-7 regulates PSA expression is not clear. Here we identify proteins that interact with CLDN-7 and determine whether such proteins can regulate PSA expression in a pattern similar to that of CLDN-7. METHODS: Our previous studies have demonstrated that in prostate cells, PSA can be regulated by a membrane protein, CLDN-7. It is important to identify the proteins that associate with CLDN-7 in its pathway of regulating PSA expression, because it is very unlikely that CLDN-7 can directly regulate PSA expression in the nucleus. To identify potential proteins that may directly interact with CLDN-7, we studied proteins that can interact with claudins. RESULTS: We found that CLDN-7 interacts with the junctional adhesion molecule A (JAM-A), which is expressed in the prostate cancer cell line, LNCaP, which expresses PSA, but not the PSA-negative prostate cell line, DU145. JAM-A regulates the expression of the prostate-specific antigen in LNCaP cells in a pattern similar to CLDN-7. CONCLUSIONS: Our results suggest that JAM-A associates with CLDN-7 and it is a component in the pathway by which CLDN-7 regulates the expression of PSA.

Gene silencing in androgen-responsive prostate cancer cells from the tissue-specific prostate-specific antigen promoter.

The success of gene therapy using a RNA interference approach relies on small interfering RNA (siRNA) expression from a highly tissue-specific RNA polymerase II promoter rather than from ubiquitous RNA polymerase III. Accordingly, we have developed a prostate-specific vector that expresses siRNAs from the human prostate-specific antigen promoter, a RNA polymerase II promoter. Our data demonstrate androgen-dependent and tissue-specific siRNA-mediated gene silencing in the androgen-responsive prostate cancer cell line, LNCaP. The biological significance was evidenced by altered apoptotic activity through the inhibition of the apoptosis-related regulatory gene. These results demonstrate that siRNA-mediated gene silencing from a tissue-specific RNA polymerase II promoter could be a potential tool for tissue-specific gene therapy.

Regression of prostate cancer xenografts by a lentiviral vector specifically expressing diphtheria toxin A.

We have constructed a prostate-specific lentiviral vector based on the promoter of the prostate-specific antigen (PSA). The PSA promoter-based lentiviral vector has been used to deliver the diphtheria toxin A (DTA) gene into prostate cancer cells, and has shown promising tissue-specific eradication of prostate cancer cells in cell culture. To evaluate the efficacy of eradicating human prostate cancer cells in vivo, we used human LNCaP prostate xenografts in nude mice as an animal model and found that with a single injection of the DTA lentiviral vector into LNCaP prostate tumors, approximately 75% of the tumors (from three experiments; conducted 9/11, 11/15 and 3/4) in the animals were completely eradicated. The DTA vector has also shown the ability to cause tumor regression in recurrent prostate tumors. Intravenous injection of the DTA lentiviral vector into nude mice elicited no pathogenic effects, suggesting that this prostate tissue-specific vector is safe for eradicating prostate cancer cells in vivo.