The anti-apoptotic protein BCL2L1/Bcl-xL is neutralized by pro-apoptotic PMAIP1/Noxa in neuroblastoma, thereby determining bortezomib sensitivity independent of prosurvival MCL1 expression.

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.

p16INK4A sensitizes human leukemia cells to FAS- and glucocorticoid-induced apoptosis via induction of BBC3/Puma and repression of MCL1 and BCL2.

Loss of CDKN2A/p16(INK4A) in hematopoietic stem cells is associated with enhanced self-renewal capacity and might facilitate progression of damaged stem cells into pre-cancerous cells that give rise to leukemia. This is also reflected by the frequent loss of the INK4A locus in acute lymphoblastic T-cell leukemia. T-cell acute lymphoblastic leukemia cells designed to conditionally express p16(INK4A) arrest in the G(0)/G(1) phase of the cell cycle and show increased sensitivity to glucocorticoid- and tumor necrosis factor receptor superfamily 6-induced apoptosis. To investigate the underlying molecular mechanism for increased death sensitivity, we interfered with specific steps of apoptosis signaling by expression of anti-apoptotic proteins. We found that alterations in cell death susceptibility resulted from changes in the composition of pro- and anti-apoptotic BCL2 proteins, i.e. repression of MCL1, BCL2, and PMAIP1/Noxa and the induction of pro-apoptotic BBC3/Puma. Interference with Puma induction by short hairpin RNA technology or retroviral expression of MCL1 or BCL2 significantly reduced both glucocorticoid- and FAS-induced cell death in p16(INK4A)-reconstituted leukemia cells. These results suggest that Puma, in concert with MCL1 and BCL2 repression, critically mediates p16(INK4A)-induced death sensitization and that in human T-cell leukemia the deletion of p16(INK4A) confers apoptosis resistance by shifting the balance of pro- and anti-apoptotic BCL2 proteins toward apoptosis protection.

Expression of coxsackie-adenovirus receptor is related to estrogen sensitivity in breast cancer.

This study analyzes the relationship between coxsackie-adenovirus receptor (CAR) expression (transmembrane and soluble isoforms) and hormone sensitivity in 95 breast cancers. Furthermore, prognostic significance of the expression of the various CAR isoforms was investigated. In addition, inducibility of CAR expression by estradiol and tamoxifen was assessed in various breast cancer cell lines. Expression of transmembrane CAR (hCAR) highly correlated with estrogen receptivity, but was independent of the expression of progesterone receptor (PR). Furthermore, hCAR expression was significantly higher in tumors with low-grade malignancy. However, no relationship between hCAR expression and tumor size, lymph node status, or survival was revealed. In the hormone receptor-positive breast cancer cell line T47-D expression of hCAR and its soluble isoforms was increased by treatment with estradiol and tamoxifen. In contrast, no induction of either CAR isoform was achieved in receptor-negative cell lines. Furthermore, enhancement of hCAR expression was significantly greater when cells were treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) than when treated with estradiol or tamoxifen. Moreover, sensitivity to TSA induction of hCAR was considerably greater in receptor-positive than in receptor-negative cell lines. No additive effect on CAR expression was found when TSA was combined with either estradiol or tamoxifen. In conclusion, the so far undescribed association between estrogen receptivity and the expression of hCAR in breast cancer seems to not only reflect a phenotype of low malignancy, but expression of hCAR may also be directly influenced by ER-specific ligands.

Repression of BIRC5/survivin by FOXO3/FKHRL1 sensitizes human neuroblastoma cells to DNA damage-induced apoptosis.

The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway regulates survival and chemotherapy resistance of neuronal cells, and its deregulation in neuroblastoma (NB) tumors predicts an adverse clinical outcome. Here, we show that inhibition of PI3K-PKB signaling in human NB cells induces nuclear translocation of FOXO3/FKHRL1, represses the prosurvival protein BIRC5/Survivin, and sensitizes to DNA-damaging agents. To specifically address whether FKHRL1 contributes to Survivin regulation, we introduced a 4-hydroxy-tamoxifen-regulated FKHRL1(A3)ERtm allele into NB cells. Conditional FKHRL1 activation repressed Survivin transcription and protein expression. Transgenic Survivin exerted a significant antiapoptotic effect and prevented the accumulation of Bim and Bax at mitochondria, the loss of mitochondrial membrane potential as well as the release of cytochrome c during FKHRL1-induced apoptosis. In concordance, Survivin knockdown by retroviral short hairpin RNA technology accelerated FKHRL1-induced apoptosis. Low-dose activation of FKHRL1 sensitized to the DNA-damaging agents doxorubicin and etoposide, whereas the overexpression of Survivin diminished FKHRL1 sensitization to these drugs. These results suggest that repression of Survivin by FKHRL1 facilitates FKHRL1-induced apoptosis and sensitizes to cell death induced by DNA-damaging agents, which supports the central role of PI3K-PKB-FKHRL1 signaling in drug resistance of human NB.