Overexpression of OATP1B3 confers apoptotic resistance in colon cancer.

Organic anion transporting polypeptide 1B3 (OATP1B3, SLCO1B3) is normally expressed in hepatocytes. In this study, we showed frequent overexpression of OATP1B3 in colorectal adenocarcinomas. Quantitative reverse transcription-PCR analysis of 17 colon tumors indicated tumoral overexpression of OATP1B3 by approximately 100-fold, compared with 20 normal colon samples (P < 0.0001). Using immunohistochemistry on a tissue microarray containing 93 evaluable colon tumor specimens, we detected immunostaining of OATP1B3 in 75 colon adenocarcinomas (81%) and no immunostaining in normal samples. To determine the functional effects of OATP1B3 expression on drug-induced apoptosis, we used camptothecin and oxaliplatin on a panel of colorectal cancer cell lines stably overexpressing OATP1B3. The results indicated that OATP1B3 overexpression enhanced cell survival in RKO, HCT-8, and HCT116(p53+/+) cells that harbor wild-type p53 but not in Caco-2 and HCT116(p53-/-) cells that lack p53, compared with the respective empty vector controls (P < 0.01). The terminal deoxynucleotidyl transferase-mediated nick-end labeling assay confirmed that HCT116(p53+/+) cells overexpressing OATP1B3 had significantly lower apoptotic levels compared with empty vector control (P < 0.001). The overexpression of OATP1B3 reduced the transcriptional activity of p53, with subsequent reductions in transcript and protein levels of its downstream transcription targets (P21WAF1 and PUMA). Overexpression of a point mutation (G583E) variant of OATP1B3 lacking transport activity did not confer an antiapoptotic effect or affect p53 transcriptional activity, suggesting that the antiapoptotic effect of OATP1B3 may be associated with its transport activity. Taken together, our results suggest that OATP1B3 overexpression in colorectal cancer cells may provide a survival advantage by altering p53-dependent pathways.

Nuclear and cytoplasmic degradation of endogenous p53 and HDM2 occurs during down-regulation of the p53 response after multiple types of DNA damage.

The principal regulator of p53 stability is HDM2, an E3 ligase mediating p53 degradation via the ubiquitin-26S proteasome pathway. Until recently, the accepted model held that p53 degradation occurs exclusively on cytoplasmic proteasomes, with an absolute requirement for nuclear export of p53 via the CRM1 pathway. However, 26S proteasomes are abundant in cytosol and nucleus. Using forced overexpression of HDM2 in mutant p53 tumor cells, we previously found that p53 degradation occurs in both the nucleus and the cytoplasm. p53 null cells coexpressing export-defective p53 and HDM2 retained partial competence for p53 degradation, challenging the obligatory export model. Because the ability of local nuclear destruction might add important control in switching off the p53 pathway, we now test this notion for physiological situations in untransfected cells and determine the significance of this regulation. Despite nuclear export blockade by leptomycin B and HTLV1-Rex protein, two potent CRM1 inhibitors, nuclear degradation of endogenous wild-type p53 and HDM2 occurs during down-regulation of the p53 response. This was seen in RKO and U2OS cells recovering from all major forms of DNA damage, including UV, gamma-IR, camptothecin, or cisplatinum. Moreover, significant nuclear degradation of endogenous p53 and HDM2 occurs in isolated nuclear fractions prepared from these recovering cells. Furthermore, nuclear proteasomes efficiently degrade ubiquitinated p53 in vitro. Our data indicate that in nonlethal outcomes of cellular stress, when DNA damage has been successfully repaired and the active p53 response needs to be down-regulated quickly to resume normal homeostasis, both nuclear and cytoplasmic proteasomes are recruited to efficiently degrade the elevated p53 and HDM2 protein levels. The physiological significance of local nuclear destruction lies in the fact that it adds tighter control and speed to switching the p53 pathway off.

DeltaNp73, a dominant-negative inhibitor of wild-type p53 and TAp73, is up-regulated in human tumors.

p73 has significant homology to p53. However, tumor-associated up-regulation of p73 and genetic data from human tumors and p73-deficient mice exclude a classical Knudson-type tumor suppressor role. We report that the human TP73 gene generates an NH(2) terminally truncated isoform. DeltaNp73 derives from an alternative promoter in intron 3 and lacks the transactivation domain of full-length TAp73. DeltaNp73 is frequently overexpressed in a variety of human cancers, but not in normal tissues. DeltaNp73 acts as a potent transdominant inhibitor of wild-type p53 and transactivation-competent TAp73. DeltaNp73 efficiently counteracts transactivation function, apoptosis, and growth suppression mediated by wild-type p53 and TAp73, and confers drug resistance to wild-type p53 harboring tumor cells. Conversely, down-regulation of endogenous DeltaNp73 levels by antisense methods alleviates its suppressive action and enhances p53- and TAp73-mediated apoptosis. DeltaNp73 is complexed with wild-type p53, as demonstrated by coimmunoprecipitation from cultured cells and primary tumors. Thus, DeltaNp73 mediates a novel inactivation mechanism of p53 and TAp73 via a dominant-negative family network. Deregulated expression of DeltaNp73 can bestow oncogenic activity upon the TP73 gene by functionally inactivating the suppressor action of p53 and TAp73. This trait might be selected for in human cancers.

Nuclear degradation of p53 occurs during down-regulation of the p53 response after DNA damage.

The principal regulator of p53 stability is HDM2, an E3 ligase that mediates p53 degradation via the ubiquitin-26S proteasome pathway. The current model holds that p53 degradation occurs exclusively on cytoplasmic proteasomes and hence has an absolute requirement for nuclear export of p53 via the CRM-1 pathway. However, proteasomes are abundant in both cytosol and nucleus, and no studies have been done to determine under what physiological circumstances p53 degradation might occur in the nucleus. We analyzed HDM2-mediated degradation of endogenous p53 in the presence of various nuclear export inhibitors of CRM-1, including leptomycin B (LMB), a noncompetitive, specific, and fast-acting inhibitor; and HTLV1-Rex protein, a potent competitive inhibitor. We found that significant HDM2-mediated p53 degradation took place in the presence of LMB or HTLV1-Rex, indicating that endogenous p53 degradation occurs locally in the nucleus, in parallel to cytoplasmic degradation. Moreover, p53 null cells that coexpressed export-defective mutants of p53 and HDM2 retained partial competence for p53 degradation. It is important that nuclear degradation of p53 occurred during the poststress recovery phase of a p53 response, after DNA damage ceased. We propose that the capability of local p53 degradation within the nucleus provides a tighter and faster control during the down-regulatory phase, when an active p53 program needs to be turned off quickly.

Disrupting the p53-mdm2 interaction as a potential therapeutic modality.

P53 and mdm2 are linked to each other through a negative feedback loop. P53 transactivates mdm2, but mdm2, in turn, is a major opponent of p53. Mdm2 promotes p53 degradation through a ubiquitin-dependent pathway on 26S proteasomes and is thought to be largely responsible for the very low levels of p53 protein in unstressed cells. The rationale for targeting the p53-mdm2 interaction therapeutically lies in the ability to activate p53 in all those tumors that retain wild type p53. Copyright 2000 Harcourt Publishers Ltd.