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FKBPL has been implicated in processes associated with cancer, including regulation of tumor growth and angiogenesis with high levels of FKBPL prognosticating for improved patient survival. Understanding how FKBPL levels are controlled within the cell is therefore critical. We have identified a novel role for RBCK1 as an FKBPL-interacting protein, which regulates FKBPL stability at the post-translational level via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-β-estradiol and interact within heat shock protein 90 chaperone complexes, together with estrogen receptor-α (ERα). Furthermore, FKBPL and RBCK1 associate with ERα at the promoter of the estrogen responsive gene, pS2, and regulate pS2 levels. MCF-7 clones stably overexpressing RBCK1 were shown to have reduced proliferation and increased levels of FKBPL and p21. Furthermore, these clones were resistant to tamoxifen therapy, suggesting that RBCK1 could be a predictive marker of response to endocrine therapy. RBCK1 knockdown using targeted small interfering RNA resulted in increased proliferation and increased sensitivity to tamoxifen treatment. Moreover, in support of our in vitro data, analysis of mRNA microarray data sets demonstrated that high levels of FKBPL and RBCK1 correlated with increased patient survival, whereas high RBCK1 predicted for a poor response to tamoxifen. Our findings support a role for RBCK1 in the regulation of FKBPL with important implications for estrogen receptor signaling, cell proliferation and response to endocrine therapy.

Drug metabolizing transgene products, which activate bioreductive cytotoxins, can be used to target treatment-resistant hypoxic tumors. The prodrug AQ4N is bioreduced in hypoxic cells by cytochrome P450s (CYPs) to the cytotoxin AQ4. Previously we have shown that intra-tumoral injection of CYP3A4 and CYP2B6 transgenes with AQ4N and radiation inhibits tumor growth. Here we examine the ability of other CYPs, in particular CYP1A1, to metabolize AQ4N, and to enhance radiosensitization. Metabolism of AQ4N was assessed using microsomes prepared from baculovirus-infected cells transfected with various CYP isoforms. AQ4N metabolism was most efficient with CYP1A1 (66.7 nmol/min/pmol) and 2B6 (34.4 nmol/min/pmol). Transient transfection of human CYP1A1+/-CYP reductase (CYPRED) was investigated in hypoxic RIF-1 mouse cells in vitro using the alkaline comet assay. There was a significant increase in DNA damage following transient transfection of CYP1A1 compared to non-transfected cells; inclusion of CYPRED provided no additional effect. In vivo, a single intra-tumoral injection of a CYP1A1 construct in combination with AQ4N (100 mg/kg i.p.) and 20 Gy X-rays caused a 16-day delay in tumor regrowth compared to tumors receiving AQ4N plus radiation and empty vector (P=0.0344). The results show the efficacy of a CYP1A1-mediated GDEPT strategy for bioreduction of AQ4N.

The bgl operon of Escherichia coli, which encodes the genes necessary for transport and catabolism of beta-glucosides, is silent in wild-type cells and is activated by the transposition of IS elements. The silent form of the operon appears to be the stable state. We isolated Bgl(-) revertants of an activated strain after growth under nonselective conditions to understand whether activation of the cryptic operon by IS elements is reversible. Genetic and molecular analyses revealed that a majority of revertants carry deletions of the bgl structural genes, indicating that an irreversible alteration has occurred in the operon. Implications of these results for the evolution and maintenance of cryptic genes are discussed.

Approximately 40% of patients with oestrogen receptor (ER)-positive breast cancers do not respond to endocrine therapies; furthermore, most responsive tumours eventually become resistant. We have identified a novel oestrogen-responsive Hsp90 co-chaperone and immunophilin, FKBPL, which affects the stability and signalling of ER with implications for breast cancer growth and sensitivity to endocrine therapies. MCF7 cells stably overexpressing FKBPL demonstrate a slower rate of proliferation and become highly dependent on oestrogen for their growth. This dependence on oestrogen renders these cells dramatically more sensitive to tamoxifen and fulvestrant. FKBPL overexpressing cells also exhibit decreased levels of ER and an oestrogen-responsive gene, cathepsin D, critical for breast cancer growth, survival and invasion. Moreover, knockdown of FKBPL using a targeted siRNA approach dramatically increases both ER and cathepsin D protein levels and cell resistance to tamoxifen. FKBPL has been previously implicated in the stabilisation of the cyclin-dependent kinase inhibitor, p21 [1]. Loss of p21 has been associated with a tamoxifen growth-inducing phenotype and hyperphosphorylation of ER at Ser118, with increased expression of ER-regulated genes. Following FKBPL knockdown, we have observed a fall in p21 levels and subsequent increase in Ser118 phosphorylation following treatment with 17β-estradiol or tamoxifen while FKBPL overexpressing cells exhibit the reverse effects. Our in vitro data support a model in which high levels of FKBPL would stabilise p21, decrease ER phosphorylation and abrogate tamoxifen-induced agonist potency, thereby increasing drug sensitivity, and suggest that FKBPL may have prognostic value that might impact upon tumour proliferative capacity and improve patient outcome. In addition, analysis of two publically available breast cancer microarray patient cohorts demonstrated that high FKBPL expression was correlated with increased overall and distant metastasis-free survival.