IKKβ Overexpression Is Correlated With P21～(CIP1/WAF1) Expression In Breast Cancers

p21^CIP1/WAF1(p21) owns duality in the regulation of oncogenesis and tumordevelpment. On the one hand, in the nuclear p21 protein binds to CDK complexes and proliferating cell nuclear antigen (PCNA), leading to cell growth arrest and DNA synthesis inhibition. On the other hand, p21 can be located in the cytoplasm where it antagonizes apoptosis, promotes tumor invasion and metastasis by binding to and inactivating tumor supressing proteins such as the apoptosis signal-regulating kinase 1 (ASK1), caspase-3 and Rho kinase, etc. Cytoplasmic p21 has been verified as a poor prognostic factor of human primary breast carcinomas.A catalytic subunit of the IKK(IκB kinase) complex, and activated by cytokines, infecition, etc, IKKβ phosphorylates and degrades IκBα, one of its substrates, and thus activating nuclear factor kappa B (NF-κB), which is a transcriptional activator of many proapoptotic, tumorigenic or angiogenic genes. Moreover, it has been found recently that IKKβ can phosphorylate Foxo3A and inhibit the tumor suppressing function of the latter. As a result, IKKβ plays an important role in promoting cell survival, tumor progression and even the development of drug resistance in cancer cells. As a matter of fact, constitutive activation of IKK has been reported in various human primary cancers and cancer cell lines, including pancreatic cancers, hematopoietic malignancies, as well as breast cancers. Accordingly, IKKβ has been recognized as a potential target for cancer treatment. Great progress has been achieved in the development of various IKKβ inhibitors.In an attempt to further characterize the function of IKKβ in cancer development and progression, we investigated the association between IKKβ and other important molecules involved in cell proliferation, cell cycle regulation, and antiapoptotic process by retrospective studies of the immunohistochemical profiles of human breastcarcinoma specimens. Interestingly, the results showed that the expression of IKKβ was positively correlated with total p21 as well as cytoplasmic p21 protein levels. In view of the report that in breast cancer cell lines, the cytokine TNFα, upstream of IKKβ, was able to induce p21 protein which was apoptotic in that experiment, we further studied whether in breast cancer cell lines IKKβ might upregulate total and subcellular p21 protein levels, and the possible mechanisms. The project has been carried out in two parts.Part 1 Association and causal relationship between IKKβ and p21 protein expression in human breast cancersObjectives: To study association among IKKβ, total and subcellular p21 protein expression in breast cancer tissues by immunohistochemistry (IHC) and analyse possible causal relationship between the two proteins by cell lines.Methods: A retrospective study was carried out using the existed IHC profiles of 128 primary breast cancer samples. IHC was performed using avidin-biodin complex technique. IKKβ immunoreactivity and p21 expression in different locations were graded according to the percentage of positively stained tumor cells, and their association was evaluated by the Pearson correlation test. On the other hand, breast cancer cell lines MCF-7 and MDA-MB-453 were stably transfected with pCDNA3 vectors (MCF-7 v2/MDA-MB-453 v1) or flag-tagged IKKβ (MCF-7 β5 and β12/MDA-MB-453 β16 and β20). p21 protein level was analyzed by Western blot using total cell lysates of these stable cell lines and those of the wild type and IKKβ knockout mouse embryo fibroblasts (MEFs ). p21 protein level was also examined using the cytoplasmic and nuclear fractionates prepared from the stable cell lines.Results: Immunohistochemical results showed that IKKβ level was associated with total p21 expression (P=0.006), cytoplasmic p21 expression (P=0.001), but not with nuclear p21 expression (P=0.309). Western blot revealed that total p21 level was obviously higher in the IKKβ transfectants than in the pCDNA3 transfectants. Similarly, total p21 expression was higher in wild type than in IKKβ-/- MEFs. Comparing the IKKβ transfectants and the vector controls, p21 expression was significantly increased in the cytoplasmic fractions but not in the nuclear fractions.Conclusion: IKKβ overexpression is associated with both increased total andcytoplasmic p21 protein levels in human breast cancer tissues. In cell lines overexpressing IKKβ, elevates total and cytoplasmic p21 levels were detected, indicating that IKKβ is the cause for total and cytoplasmic p21 upregulation.Part 2 Mechanism studies for IKKβ -mediated total and cytoplasmic p21elevationObjectives: Firstly to investigate whether IKKβ elevates total p21 protein level by influencing p21 protein stability, mRNA level, as well as promoter transcription. Secondly, to study whether Akt, known to cause p21 cytoplasmic translocation, is involved in IKKβ-mediated cytoplasmic p21 upregulation, and whether IKKβ may influence Akt expression.Methods: To investigate how IKKβ upregulates total p21 level, p21 protein turnover rate was examined by treating the IKKβ stable cell lines and the controls with cycloheximide and the following western blot with the anti-p21 antibody. Then p21 mRNA levels in the stable cell lines were compared by RT-PCR. Finally, transcriptional activity of the p21 promoter in the stable cell lines and the MEFs with or without IKKβ was analysed by dual luciferase assay using the WWP-Luc plasmid, a wild-type p21 promoter fused with a firefly luciferase reporter.To determine whether Akt is involved in IKKβ-mediated cytoplasmic p21 upregulation, subcellular p21 protein levels were evaluated in the MDA-MB-453 stable cell lines treated with or without the PI3K inhibitor LY294002. To find out whether IKKβ may play a role in the regulation of Akt activation, total and phosphorylated Akt (Ser 473) levels in the IKKβ stable cell lines and the controls were compared by western blot. Total and phosphorylated Akt levels were also examined in the same stable cell lines treated with or without IKK inhibitor Wedelolactone.Results: p21 protein stability was not affected by IKKβ overexpression. p21 mRNA level was higher in the IKKβ stable transfectants than in the vector controls. p21 promoter transcriptional activity was higher in IKKβ stable transfectants as well as in the wild type MEF cell line, comparing with that of the vector controls and IKKβ-/- MEFs respectively. With reduced Akt phosphorylation, LY294002treatment resulted in more dramatic downregulation of cytoplasmic p21 in the IKKβ stable transfectants than in the vector controls, as well as much less difference of cytoplasmic p21 levels in these two kinds of stable transfectants. Phorsphorylated Akt rather than total Akt was upregulated in the IKKβ stable cell lines comparing with the controls. Wedelolactone treatment gave rise to downregulation of Akt phosphorylation instead of total Akt level.Conclusions: IKKβ may upregulate total p21 protein level through elevating p21 mRNA level and activating p21 transcription. Akt is involved in IKKβ mediated cytoplasmic p21 elevation. IKKβ overexpression may increase cytoplasmic p21 expression via upregulation of phosphorylated Akt. IKKβ upregulates Akt phosphorylation without elevation of total Akt protein.