Stavroula Baritaki

Polycomb Protein EZH2 Regulates Tumor Invasion via the Transcriptional Repression of the Metastasis Suppressor RKIP in Breast and Prostate Cancer

Epigenetic modifications such as histone methylation play an important role in human cancer metastasis. Enhancer of zeste homolog 2 (EZH2), which encodes the histone methyltransferase component of the polycomb repressive complex 2 (PRC2), is overexpressed widely in breast and prostate cancers and epigenetically silences tumor suppressor genes. Expression levels of the novel tumor and metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) have been shown to correlate negatively with those of EZH2 in breast and prostate cell lines as well as in clinical cancer tissues. Here, we show that the RKIP/EZH2 ratio significantly decreases with the severity of disease and is negatively associated with relapse-free survival in breast cancer. Using a combination of loss- and gain-of-function approaches, we found that EZH2 negatively regulated RKIP transcription through repression-associated histone modifications. Direct recruitment of EZH2 and suppressor of zeste 12 (Suz12) to the proximal E-boxes of the RKIP promoter was accompanied by H3-K27-me3 and H3-K9-me3 modifications. The repressing activity of EZH2 on RKIP expression was dependent on histone deacetylase promoter recruitment and was negatively regulated upstream by miR-101. Together, our findings indicate that EZH2 accelerates cancer cell invasion, in part, via RKIP inhibition. These data also implicate EZH2 in the regulation of RKIP transcription, suggesting a potential mechanism by which EZH2 promotes tumor progression and metastasis.

Chemotherapeutic drugs sensitize cancer cells to TRAIL-mediated apoptosis: up-regulation of DR5 and inhibition of Yin Yang 1

Several chemotherapeutic drugs in combination with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) result in reversal of resistance to TRAIL-mediated apoptosis through up-regulation of DR5 expression. The promoter of DR5 has one putative binding site for the transcription repressor Yin Yang 1 (YY1), and thus, we hypothesized that the sensitizing drugs may inhibit YY1. We have found that treatment of tumor cells with various chemotherapeutic drugs inhibited nuclear factor-κB. We examined whether drugs also inhibit YY1 activity and whether YY1 inhibition correlates with up-regulation of DR5 expression and sensitization of cells to TRAIL-induced apoptosis. The TRAIL- and drug-resistant prostate carcinoma PC-3 cell line was treated with CDDP, VP-16, ADR, and vincristine. DR5 luciferase reporter constructs and small interfering RNA against YY1 were used to determine the role of YY1 in DR5 transcription. Pretreatment of PC-3 cells and other tumor cell lines with various chemotherapeutic drugs sensitized the cells to TRAIL-induced apoptosis concurrently with up-regulation of DR5 expression and inhibition of YY1 expression and its DNA-binding activity. The baseline luciferase activity in PC-3 cells transfected with the wild-type DR5 reporter was significantly augmented in cells transfected with DR5 constructs carrying deletions or mutation in the YY1-binding site. Treatment with drug enhanced DR5 wild-type luciferase activity, with no increase in cells transfected with the YY1-deleted or YY1-mutated constructs. Cells transfected with YY1 small interfering RNA showed up-regulation of DR5 expression and sensitization to TRAIL-mediated apoptosis. The findings provide evidence that drug-induced sensitization of tumor cells to TRAIL is mediated, in part, by inhibition of the transcription repressor YY1 and up-regulation of DR5 expression. Hence, YY1 may be a potential therapeutic target to reverse resistance to TRAIL-induced apoptosis. [Mol Cancer Ther 2007;6(4):1387–99]

Regulation of Tumor Cell Sensitivity to TRAIL-Induced Apoptosis by the Metastatic Suppressor Raf Kinase Inhibitor Protein via Yin Yang 1 Inhibition and Death Receptor 5 Up-Regulation

Raf-1 kinase inhibitor protein (RKIP) has been implicated in the regulation of cell survival pathways and metastases, and is poorly expressed in tumors. We have reported that the NF-κB pathway regulates tumor resistance to apoptosis by the TNF-α family via inactivation of the transcription repressor Yin Yang 1 (YY1). We hypothesized that RKIP overexpression may regulate tumor sensitivity to death ligands via inhibition of YY1 and up-regulation of death receptors (DRs). The TRAIL-resistant prostate carcinoma PC-3 and melanoma M202 cell lines were examined. Transfection with CMV-RKIP, but not with control CMV-EV, sensitized the cells to TRAIL-mediated apoptosis. Treatment with RKIP small interfering RNA (siRNA) inhibited TRAIL-induced apoptosis. RKIP overexpression was paralleled with up-regulation of DR5 transcription and expression; no change in DR4, decoy receptor 1, and decoy receptor 2 expression; and inhibition of YY1 transcription and expression. Inhibition of YY1 by YY1 siRNA sensitized the cells to TRAIL apoptosis concomitantly with DR5 up-regulation. RKIP overexpression inhibited several antiapoptotic gene products such as X-linked inhibitor of apoptosis (XIAP), c-FLIP long, and Bcl-xL that were accompanied with mitochondrial membrane depolarization. RKIP overexpression in combination with TRAIL resulted in the potentiation of these above effects and activation of caspases 8, 9, and 3, resulting in apoptosis. These findings demonstrate that RKIP overexpression regulates tumor cell sensitivity to TRAIL via inhibition of YY1, up-regulation of DR5, and modulation of apoptotic pathways. We suggest that RKIP may serve as an immune surveillance cancer gene, and its low expression or absence in tumors allows the tumor to escape host immune cytotoxic effector cells.

The Role of B-RAF Mutations in Melanoma and the Induction of EMT via Dysregulation of the NF-κB/Snail/RKIP/PTEN Circuit

Melanoma is a highly metastatic cancer, and there are no current therapeutic modalities to treat this deadly malignant disease once it has metastasized. Melanoma cancers exhibit B-RAF mutations in up to 70% of cases. B-RAF mutations are responsible, in large part, for the constitutive hyperactivation of survival/antiapoptotic pathways such as the MAPK, NF-κB, and PI3K/AKT. These hyperactivated pathways regulate the expression of genes targeting the initiation of the metastatic cascade, namely, the epithelial to mesenchymal transition (EMT). EMT is the result of the expression of mesenchymal gene products such as fibronectin, vimentin, and metalloproteinases and the invasion and inhibition of E-cadherin. The above pathways cross-talk and regulate each others activities and functions. For instance, the NF-κB pathway directly regulates EMT through the transcription of gene products involved in EMT and indirectly through the transcriptional up-regulation of the metastasis inducer Snail. Snail, in turn, suppresses the expression of the metastasis suppressor gene product Raf kinase inhibitor protein RKIP (inhibits the MAPK and the NF-κB pathways) as well as PTEN (inhibits the PI3K/AKT pathway). The role of B-RAF mutations in melanoma and their direct role in the induction of EMT are not clear. This review discusses the hypothesis that B-RAF mutations are involved in the dysregulation of the NF-κB/Snail/RKIP/PTEN circuit and in both the induction of EMT and metastasis. The therapeutic implications of the dysregulation of the above circuit by B-RAF mutations are such that they offer novel targets for therapeutic interventions in the treatment of EMT and metastasis.

Pivotal Roles of Snail Inhibition and RKIP Induction by the Proteasome Inhibitor NPI-0052 in Tumor Cell Chemoimmunosensitization

The novel proteasome inhibitor NPI-0052 has been shown to sensitize tumor cells to apoptosis by various chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although the mechanisms involved are not clear. We hypothesized that NPI-0052-mediated sensitization may result from NF-kappaB inhibition and downstream modulation of the metastasis inducer Snail and the metastasis suppressor/immunosurveillance cancer gene product Raf-1 kinase inhibitory protein (RKIP). Human prostate cancer cell lines were used as models, as they express different levels of these proteins. We show that NPI-0052 inhibits both NF-kappaB and Snail and induces RKIP expression, thus resulting in cell sensitization to CDDP and TRAIL. The direct role of NF-kappaB inhibition in sensitization was corroborated with the NF-kappaB inhibitor DHMEQ, which mimicked NPI-0052 in sensitization and inhibition of Snail and induction of RKIP. The direct role of Snail inhibition by NPI-0052 in sensitization was shown with Snail small interfering RNA, which reversed resistance and induced RKIP. Likewise, the direct role of RKIP induction in sensitization was revealed by both overexpression of RKIP (mimicking NPI-0052) and RKIP small interfering RNA that inhibited NPI-0052-mediated sensitization. These findings show that NPI-0052 modifies the NF-kappaB-Snail-RKIP circuitry in tumor cells and results in downstream inhibition of antiapoptotic gene products and chemoimmunosensitization. The findings also identified Snail and RKIP as targets for reversal of resistance.

Dual role of NO donors in the reversal of tumor cell resistance and EMT: Downregulation of the NF-κB/Snail/YY1/RKIP circuitry.

Several studies have implicated the role of Nitric Oxide (NO) in the regulation of tumor cell behavior and have shown that NO either promotes or inhibits tumorigenesis. These conflicting findings have been resolved, in part, by the levels of NO used such that low levels promote tumor growth and high levels inhibit tumor growth. Our studies have focused on the use of high levels of NO provided primarily by the NO donor, DETANONOate. We have shown that treatment of resistant tumor cells with DETANONOate sensitizes them to apoptosis by both chemotherapeutic drugs and cytotoxic immunotherapeutic ligands. The underlying mechanisms by which NO sensitizes tumor cells to apoptosis were shown to be regulated, in part, by NO-mediated inhibition of the NF-κB survival/anti-apoptotic pathways and downstream of NF-κB by inhibition of the transcription factor Yin Yang 1 (YY1). In addition to NO-induced sensitization to apoptosis, we have also shown that NO induced the expression of the metastasis-suppressor/immunosurveillance cancer gene product, Raf-1 kinase inhibitor protein (RKIP). Overexpression of RKIP mimics NO in tumor cells-induced sensitization to apoptosis. The induction of RKIP by NO was the result of the inhibition of the RKIP repressor, Snail, downstream of NF-κB. These findings established the presence of a dysregulated NF-κB/Snail/YY1/ RKIP circuitry in resistance and that treatment with NO modifies this loop in tumor cells in favor of the inhibition of tumor cell survival and the response to cytotoxic drugs. Noteworthy, the NF-κB/Snail/YY1/RKIP loop consists of gene products that regulate the epithelial to mesenchymal transition (EMT) and, thus, tumor metastasis. Hence, we have found that treatment of metastatic cancer cell lines with DETANONOate inhibited the EMT phenotype, through both the inhibition of the metastasis-inducers, NF-κB and Snail and the induction of the metastasis-suppressor, RKIP. Altogether, the above findings establish, for the first time, the dual role of high levels of NO in the sensitization of tumor cells to apoptotic stimuli as well as inhibition of EMT. Hence, NO donors may be considered as novel potential therapeutic agents with dual roles in the treatment of patients with refractory cancer and in the prevention of the initiation of the metastatic cascade via EMT.

Inhibition of Yin Yang 1-Dependent Repressor Activity of DR5 Transcription and Expression by the Novel Proteasome Inhibitor NPI-0052 Contributes to its TRAIL-Enhanced Apoptosis in Cancer Cells

TRAIL promotes apoptotic tumor cell death; however, TRAIL-resistant tumors need to be sensitized to reverse resistance. Proteasome inhibitors potentiate TRAIL apoptosis in vitro and in vivo and correlate with up-regulation of death receptor 5 (DR5) via an unknown mechanism. We hypothesized that the proteasome inhibitor NPI-0052 inhibits the transcription repressor Yin Yang 1 (YY1) which regulates TRAIL resistance and negatively regulates DR5 transcription. Treatment of PC-3 and Ramos cells with NPI-0052 (≤2.5 nM) and TRAIL sensitizes the tumor cells to TRAIL-induced apoptosis. By comparison to bortezomib, a 400-fold less concentration of NPI-0052 was used. NPI-0052 up-regulated DR5 reporter activity and both surface and total DR5 protein expression. NPI-0052-induced inhibition of NF-κB activity was involved in TRAIL sensitization as corroborated by the use of the NF-κB inhibitor dehydroxymethylepoxyquinomicin. NPI-0052 inhibited YY1 promoter activity as well as both YY1 mRNA and protein expression. The direct role of NPI-0052-induced inhibition of YY1 and up-regulation of DR5 in the regulation of TRAIL sensitivity was demonstrated by the use of YY1 small interfering RNA. The NPI-0052-induced sensitization to TRAIL involved activation of the intrinsic apoptotic pathway and dysregulation of genes that regulate apoptosis. The NPI-0052 concentrations used for TRAIL sensitization were not toxic to human hematopoetic stem cells. The present findings demonstrate, for the first time, the potential mechanism by which a proteasome inhibitor, like NPI-0052, inhibits the transcription repressor YY1 involved in TRAIL resistance and DR5 regulation. The findings also suggest the therapeutic application of subtoxic NPI-0052 concentrations in combination with TRAIL/agonist DR4/DR5 mAbs in the treatment of TRAIL-resistant tumors.

Nitric oxide sensitizes tumor cells to TRAIL-induced apoptosis via inhibition of the DR5 transcription repressor Yin Yang 1

Treatment of TRAIL-resistant tumor cells with the nitric oxide donor DETANONOate sensitizes the tumor cells to TRAIL-induced apoptosis concomitantly with DR5 upregulation. The mechanism of sensitization was examined based on the hypothesis that DETANONOate inhibits a transcription repressor Yin Yang 1 (YY1) that negatively regulates DR5 transcription. Treatment of the prostate carcinoma cell lines with DETANONOate inhibited both NF-kappaB and YY1 DNA-binding activities concomitantly with upregulation of DR5 expression. The direct role of YY1 in the regulation of TRAIL resistance was demonstrated in cells treated with YY1 siRNA resulting in TRAIL-induced apoptosis. The role of YY1 in the transcriptional regulation of DR5 was examined in cells treated with a DR5 luciferase reporter system (pDR5) and two constructs, namely, the pDR5/-605 construct with a deletion of the putative YY1 DNA-binding region (-1224 to -605) and a construct pDR5-YY1 with a mutation of the YY1 DNA-binding site. A significant (3-fold) augmentation of luciferase activity over baseline transfection with pDR5 was observed in cells transfected with the modified constructs. ChIP analysis corroborated the YY1 binding to the DR5 promoter. In vivo, tissues from nude mice bearing the PC-3 xenograft and treated with DETANONOate showed inhibition of YY1 and upregulation of DR5. The present findings demonstrate that YY1 negatively regulates DR5 transcription and expression and these correlated with resistance to TRAIL-induced apoptosis. DETANONOate inhibits both NF-kappaB and YY1 and in combination with TRAIL reverses tumor cell resistance to TRAIL apoptosis.

Reversal of tumor resistance to apoptotic stimuli by alteration of membrane fluidity: therapeutic implications.

In recent years, significant development and improvement have been observed in the treatment of cancer; however, relapses and recurrences occur frequently and there have not been any current therapies to treat such cancers. Cancers resistant to conventional therapies develop several mechanisms to escape death-inducing stimuli. A poorly understood mechanism is the involvement of the cancer cell plasma membrane composition and architecture and their involvement in regulating drug-inducing stimuli leading to cell death. Although the basic structure of the biological membrane was established 80 years ago, study of the physical properties of lipid bilayers still provides significant information regarding membrane organization and dynamics. Membrane fluidity is probably the most important physicochemical property of cell membranes. Alterations of membrane fluidity can seriously affect functional properties of the cell and induction of apoptotic pathways resulting in cell death. The role of membrane fluidity in the apoptotic process is clearly exemplified as it is seriously disrupted as a result of cell injury. The molecular signaling pathways leading to apoptosis are currently promising areas of research investigation and lead to unravel the underlying molecular mechanisms of tumor cells resistance to apoptotic stimuli and hence the development of new effective therapeutic agents. Recent findings indicate that most anticancer agents induce apoptosis, directly or indirectly, through alterations of tumor cell membrane fluidity. The present chapter summarizes the relationship between alterations of tumor cell membrane fluidity and tumor cell response to apoptotic-inducing stimuli. Several potential therapeutic applications directed at tumor cell membrane fluidity are proposed.

Mechanisms of nitric oxide-mediated inhibition of EMT in cancer: Inhibition of the metastasis-inducer Snail and induction of the metastasis-suppressor RKIP

The role of nitric oxide (NO) in cancer has been controversial and is based on the levels of NO and the responsiveness of the tumor type. It remains unclear whether NO can inhibit the epithelial to mesenchymal transition (EMT) in cancer cells. EMT induction is mediated, in part, by the constitutive activation of the metastasis-inducer transcription factor, Snail and EMT can be inhibited by the metastasis-suppressor Raf-1 kinase inhibitor protein (RKIP) and E-cadherin. Snail is transcriptionally regulated by NF-κB and in turn, Snail represses RKIP transcription. Hence, we hypothesized that high levels of NO, that inhibit NF-κB activity, may also inhibit Snail and induce RKIP and leading to inhibition of EMT. We show that treatment of human prostate metastatic cell lines with the NO donor, DETANONOate, inhibits EMT and reverses both the mesenchymal phenotype and the cell invasive properties. Further, treatment with DETANONOate inhibits Snail expression and DNA-binding activity in parallel with the upregulation of RKIP and E-cadherin protein levels. The pivotal roles of Snail inhibition and RKIP induction in DETANONOate-mediated inhibition of EMT were corroborated by both Snail silencing by siRNA and by ectopic expression of RKIP. The in vitro findings were validated in vivo in mice bearing PC-3 xenografts and treated with DETANONOate. The present findings show, for the first time, the novel role of high subtoxic concentrations of NO in the inhibition of EMT. Thus, NO donors may exert therapeutic activities in the reversal of EMT and metastasis.