Targeting GLI by GANT61 involves mechanisms dependent on inhibition of both transcription and DNA licensing.

The GLI genes are transcription factors and in cancers are oncogenes, aberrantly and constitutively activated. GANT61, a specific GLI inhibitor, has induced extensive cytotoxicity in human models of colon cancer. The FOXM1 promoter was determined to be a transcriptional target of GLI1. In HT29 cells, inhibition of GLI1 binding at the GLI consensus sequence by GANT61 led to inhibited binding of Pol II, the pause-release factors DSIF, NELF and p-TEFb. The formation of R-loops (RNA:DNA hybrids, ssDNA), were reduced by GANT61 at the FOXM1 promoter. Pretreatment of HT29 cells with α-amanitin reduced GANT61-induced γH2AX foci. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication to be linked. By co-immunoprecipitation and confocal microscopy, GLI1 co-localized with the DNA licensing factors ORC4, CDT1, and MCM2. Significant co-localization of GLI1 and ORC4 was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. CDT1 was found to be a transcription target of GLI1. Overexpression of CDT1 in HT29 and SW480 cells reduced GANT61-induced cell death, gH2AX foci, and cleavage of caspase-3. Data demonstrate involvement of transcription and of DNA replication licensing factors by non-transcriptional and transcriptional mechanisms in the GLI-dependent mechanism of action of GANT61.

Mode and specificity of binding of the small molecule GANT61 to GLI determines inhibition of GLI-DNA binding.

The GLI genes, GLI1 and GLI2, are transcription factors that regulate target genes at the distal end of the canonical Hedgehog (HH) signaling pathway (SHH->PTCH->SMO->GLI), tightly regulated in embryonic development, tissue patterning and differentiation. Both GLI1 and GLI2 are oncogenes, constitutively activated in many types of human cancers. In colon cancer cells oncogenic KRAS-GLI signaling circumvents the HH-SMO-GLI axis to channel through and activate GLI in the transcriptional regulation of target genes. We have observed extensive cell death in a panel of 7 human colon carcinoma cell lines using the small molecule GLI inhibitor GANT61. Using computational docking and experimental confirmation by Surface Plasmon Resonance, GANT61 binds to the 5-zinc finger GLI1 protein between zinc fingers 2 and 3 at sites E119 and E167, independent of the GLI-DNA binding region, and conserved between GLI1 and GLI2. GANT61 does not bind to other zinc finger transcription factors (KLF4, TFIIß). Mutating the predicted GANT61 binding sites in GLI1 significantly inhibits GANT61-GLI binding and GLI-luciferase activity. Data establish the specificity of GANT61 for targeting GLI, and substantiate the critical role of GLI in cancer cell survival. Thus, targeting GLI in cancer therapeutics may be of high impact.

Hedgehog signaling regulates telomerase reverse transcriptase in human cancer cells.

The Hedgehog (HH) signaling pathway is critical for normal embryonic development, tissue patterning and cell differentiation. Aberrant HH signaling is involved in multiple human cancers. HH signaling involves a multi-protein cascade activating the GLI proteins that transcriptionally regulate HH target genes. We have previously reported that HH signaling is essential for human colon cancer cell survival and inhibition of this signal induces DNA damage and extensive cell death. Here we report that the HH/GLI axis regulates human telomerase reverse transcriptase (hTERT), which determines the replication potential of cancer cells. Suppression of GLI1/GLI2 functions by a C-terminus truncated GLI3 repressor mutant (GLI3R), or by GANT61, a pharmacological inhibitor of GLI1/GLI2, reduced hTERT protein expression in human colon cancer, prostate cancer and Glioblastoma multiforme (GBM) cell lines. Expression of an N-terminus deleted constitutively active mutant of GLI2 (GLI2ΔN) increased hTERT mRNA and protein expression and hTERT promoter driven luciferase activity in human colon cancer cells while GANT61 inhibited hTERT mRNA expression and hTERT promoter driven luciferase activity. Chromatin immunoprecipitation with GLI1 or GLI2 antibodies precipitated fragments of the hTERT promoter in human colon cancer cells, which was reduced upon exposure to GANT61. In contrast, expression of GLI1 or GLI2ΔN in non-malignant 293T cells failed to alter the levels of hTERT mRNA and protein, or hTERT promoter driven luciferase activity. Further, expression of GLI2ΔN increased the telomerase enzyme activity, which was reduced by GANT61 administration in human colon cancer, prostate cancer, and GBM cells. These results identify hTERT as a direct target of the HH signaling pathway, and reveal a previously unknown role of the HH/GLI axis in regulating the replication potential of cancer cells. These findings are of significance in understanding the important regulatory mechanisms that determine the functions of HH/GLI signaling in cancer cells.

Regulation of DNA damage following termination of Hedgehog (HH) survival signaling at the level of the GLI genes in human colon cancer.

Transcriptional regulation of the Hedgehog (HH) signaling response is mediated by GLI genes (GLI1, GLI2) downstream of SMO, that are also activated by oncogenic signaling pathways. We have demonstrated the importance of targeting GLI downstream of SMO in the induction of cell death in human colon carcinoma cells. In HT29 cells inhibition of GLI1/GLI2 by the small molecule inhibitor GANT61 induced DNA double strand breaks (DSBs) and activation of ATM, MDC1 and NBS1; γH2AX and MDC1, NBS1 and MDC1 co-localized in nuclear foci. Early activation of ATM was decreased by 24 hr, when p-NBS1(Ser343), activated by ATM, was significantly reduced in cell extracts. Bound γH2AX was detected in isolated chromatin fractions or nuclei during DNA damage but not during DNA repair. MDC1 was tightly bound to chromatin at 32 hr as cells accumulated in early S-phase prior to becoming subG1, and during DNA repair. Limited binding of NBS1 was detected at all times during DNA damage but was strongly bound during DNA repair. Transient overexpression of NBS1 protected HT29 cells from GANT61-induced cell death, while knockdown of H2AX by H2AXshRNA delayed DNA damage signaling. Data demonstrate following GLI1/GLI2 inhibition: 1) induction of DNA damage in cells that are also resistant to SMO inhibitors, 2) dynamic interactions between γH2AX, MDC1 and NBS1 in single cell nuclei and in isolated chromatin fractions, 3) expression and chromatin binding properties of key mediator proteins that mark DNA damage or DNA repair, and 4) the importance of NBS1 in the DNA damage response mechanism.

The GLI genes as the molecular switch in disrupting Hedgehog signaling in colon cancer.

The Hedgehog (HH) signaling pathway leads to activation of GLI, which transcriptionally regulate target genes. Regulated HH signaling activity is critical during embryogenesis while aberrantly activated HH signaling is evident in a variety of human cancers. Canonical HH signaling engages the transmembrane receptor Patched (PTCH) and the signaling intermediate Smoothened (SMO) to activate GLI1 and GLI2. In addition GLI1 and GLI2 are activated by non-canonical oncogenic signaling pathways to further drive HH-dependent survival. We have demonstrated in human colon carcinoma cells that inhibition of the RAS/RAF pathway by U0126 decreases p-ERK protein expression and also inhibits GLI-luciferase activity and GLI1 mRNA and protein levels. Of importance is the demonstration that targeting of SMO (using cyclopamine) has minimal effect on cell survival in comparison to the inhibition of GLI (using GANT61), which induced extensive cell death in 7/7 human colon carcinoma cell lines. Genetic inhibition of the function of GLI1 and GLI2 by transient transfection of the C-terminus deleted repressor GLI3R, reduced proliferation and induced cleavage of caspase-3 and cell death in HT29 cells, similar to the effects of GANT61. Mechanistically, downstream of GLI1 and GLI2 inhibition, γH2AX (a marker of DNA double strand breaks) expression was upregulated, and γH2AX nuclear foci were demonstrated in cells that expressed GLI3R. Activation of the ATM/Chk2 axis with co-localization of γH2AX and p-Chk2 nuclear foci were demonstrated following GLI1/GLI2 inhibition. GANT61 induced cellular accumulation at G1/S and early S with no further progression before cells became subG1, while cDNA microarray gene profiling demonstrated downregulation of genes involved in DNA replication, the DNA damage response, and DNA repair, mechanisms that are currently being pursued. These studies highlight the importance of targeting the GLI genes downstream of SMO for terminating HH-dependent survival, suggesting that GLI may constitute a molecular switch that determines the balance between cell survival and cell death in human colon carcinoma.

An antiapoptotic BCL-2 family expression index predicts the response of chronic lymphocytic leukemia to ABT-737.

The antiapoptotic BCL-2 proteins regulate lymphocyte survival and are over-expressed in lymphoid malignancies, including chronic lymphocytic leukemia. The small molecule inhibitor ABT-737 binds with high affinity to BCL-2, BCL-XL, and BCL-W but with low affinity to MCL-1, BFL-1, and BCL-B. The active analog of ABT-737, navitoclax, has shown a high therapeutic index in lymphoid malignancies; developing a predictive marker for it would be clinically valuable for patient selection or choice of drug combinations. Here we used RT-PCR as a highly sensitive and quantitative assay to compare expression of antiapoptotic BCL-2 genes that are known to be targeted by ABT-737. Our findings reveal that the relative ratio of MCL-1 and BFL-1 to BCL-2 expression provides a highly significant linear correlation with ABT-737 sensitivity (r = 0.6, P < .001). In contrast, antiapoptotic transcript levels, used individually or in combination for high or low affinity ABT-737-binding proteins, could not predict ABT-737 sensitivity. The (MCL-1 + BFL-1)/BCL-2 ratio was validated in a panel of leukemic cell lines subjected to genetic and pharmacologic manipulations. Changes after ABT-737 treatment included increased expression of BFL-1 and BCL-B that may contribute to treatment resistance. This study defines a highly significant BCL-2 expression index for predicting the response of CLL to ABT-737.

Blocking Hedgehog survival signaling at the level of the GLI genes induces DNA damage and extensive cell death in human colon carcinoma cells.

Canonical Hedgehog (HH) signaling is characterized by Smoothened (Smo)-dependent activation of the transcription factors Gli1 and Gli2, which regulate HH target genes. In human colon carcinoma cells, treatment with the Gli small-molecule inhibitor GANT61 induces extensive cell death in contrast to the Smo inhibitor cyclopamine. Here we elucidate cellular events upstream of cell death elicited by GANT61, which reveal the basis for its unique cytotoxic activity in colon carcinoma cells. Unlike cyclopamine, GANT61 induced transient cellular accumulation at G(1)-S (24 hours) and in early S-phase (32 hours), with elevated p21(Cip1), cyclin E, and cyclin A in HT29 cells. GANT61 induced DNA damage within 24 hours, with the appearance of p-ATM and p-Chk2. Pharmacologic inhibition of Gli1 and Gli2 by GANT61 or genetic inhibition by transient transfection of the Gli3 repressor (Gli3R) downregulated Gli1 and Gli2 expression and induced γH2AX, PARP cleavage, caspase-3 activation, and cell death. GANT61 induced γH2AX nuclear foci, while transient transfection of Gli3R showed expression of Gli3R and γH2AX foci within the same nuclei in HT29, SW480, and HCT116. GANT61 specifically targeted Gli1 and Gli2 substantiated by specific inhibition of (i) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2, (ii) Gli-luciferase activity, and (iii) transcriptional activation of BCL-2. Taken together, these findings establish that inhibition of HH signaling at the level of the GLI genes downstream of Smo is critical in the induction of DNA damage in early S-phase, leading to cell death in human colon carcinoma cells.

Identification of STAT3-independent regulatory effects for protein inhibitor of activated STAT3 by binding to novel transcription factors.

Protein Inhibitor of Activated Signal Transducer and Activators of Transcription 3 (PIAS3) is a molecule that regulates STAT3 and has antiproliferative properties. Glioblastoma and squamous cell lung cancer lack PIAS3 expression. To test the hypothesis that PIAS3 transcriptional effects are STAT3-independent, we developed models for STAT3 knockdown and PIAS3 over-expression. PIAS3 expression results in a distinct transcriptional profile that does not occur with STAT3 knockdown. We identify novel transcription factor binding partners for PIAS3 including ETS, EGR1, NR1I2, and GATA1. PIAS3 binds to these factors and regulates their transcriptional effects resulting in alterations in canonical pathways including Wnt/ß-catenin signaling and functions such as cell death and proliferation. A model is proposed by which PIAS3 effects EGR1 regulated pathways.

Hedgehog signaling drives cellular survival in human colon carcinoma cells.

Aberrant activation of Hedgehog (HH) signaling is implicated in many human cancers. Classical HH signaling is characterized by Smoothened (Smo)-dependent activation of Gli1 and Gli2, which transcriptionally regulate target genes. A small molecule inhibitor of Gli1 and Gli2, GANT61, was used to block HH signaling in human colon carcinoma cell lines that express HH signaling components. GANT61 administration induced robust cytotoxicity in 5 of 6 cell lines and moderate cytotoxicity in the remaining 1 cell line. In comparison, the classical Smo inhibitor, cyclopamine, induced modest cytotoxicity. Further, GANT61 treatment abolished the clonogenicity of all six human colon carcinoma cell lines. Analysis of the molecular mechanisms of GANT61-induced cytotoxicity in HT29 cells showed increased Fas expression and decreased expression of PDGFRα, which also regulates Fas. Furthermore, DR5 expression was increased whereas Bcl-2 (direct target of Gli2) was downregulated following GANT61 treatment. Suppression of Gli1 by shRNA mimicked the changes in gene expression observed in GANT61-treated cells. Overexpression of dominant-negative FADD (to abrogate Fas/DR5-mediated death receptor signaling) and/or Bcl-2 (to block mitochondria-mediated apoptosis) partially rescued GANT61-induced cytotoxicity in HT29 cells. Thus, activated GLI genes repress DR5 and Fas expressions while upregulating Bcl-2 and PDGFRα expressions to inhibit Fas and facilitate cell survival. Collectively, these results highlight the importance of Gli activation downstream of Smo as a therapeutic target in models of human colon carcinoma.

cDNA microarray gene expression profiling of hedgehog signaling pathway inhibition in human colon cancer cells.

BACKGROUND: Hedgehog (HH) signaling plays a critical role in normal cellular processes, in normal mammalian gastrointestinal development and differentiation, and in oncogenesis and maintenance of the malignant phenotype in a variety of human cancers. Increasing evidence further implicates the involvement of HH signaling in oncogenesis and metastatic behavior of colon cancers. However, genomic approaches to elucidate the role of HH signaling in cancers in general are lacking, and data derived on HH signaling in colon cancer is extremely limited. METHODOLOGY/PRINCIPAL FINDINGS: To identify unique downstream targets of the GLI genes, the transcriptional regulators of HH signaling, in the context of colon carcinoma, we employed a small molecule inhibitor of both GLI1 and GLI2, GANT61, in two human colon cancer cell lines, HT29 and GC3/c1. Cell cycle analysis demonstrated accumulation of GANT61-treated cells at the G1/S boundary. cDNA microarray gene expression profiling of 18,401 genes identified Differentially Expressed Genes (DEGs) both common and unique to HT29 and GC3/c1. Analyses using GenomeStudio (statistics), Matlab (heat map), Ingenuity (canonical pathway analysis), or by qRT-PCR, identified p21(Cip1) (CDKN1A) and p15(Ink4b) (CDKN2B), which play a role in the G1/S checkpoint, as up-regulated genes at the G1/S boundary. Genes that determine further cell cycle progression at G1/S including E2F2, CYCLIN E2 (CCNE2), CDC25A and CDK2, and genes that regulate passage of cells through G2/M (CYCLIN A2 [CCNA2], CDC25C, CYCLIN B2 [CCNB2], CDC20 and CDC2 [CDK1], were down-regulated. In addition, novel genes involved in stress response, DNA damage response, DNA replication and DNA repair were identified following inhibition of HH signaling. CONCLUSIONS/SIGNIFICANCE: This study identifies genes that are involved in HH-dependent cellular proliferation in colon cancer cells, and following its inhibition, genes that regulate cell cycle progression and events downstream of the G1/S boundary.

Inhibition of NF-kappaB signaling by quinacrine is cytotoxic to human colon carcinoma cell lines and is synergistic in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or oxaliplatin.

Colorectal cancer is the third most common malignancy in the United States. Modest advances with therapeutic approaches that include oxaliplatin (L-OHP) have brought the median survival rate to 22 months, with drug resistance remaining a significant barrier. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is undergoing clinical evaluation. Although human colon carcinomas express TRAIL receptors, they can also demonstrate TRAIL resistance. Constitutive NF-kappaB activation has been implicated in resistance to TRAIL and to cytotoxic agents. We have demonstrated constitutive NF-kappaB activation in five of six human colon carcinoma cell lines; this activation is inhibited by quinacrine. Quinacrine induced apoptosis in colon carcinomas and potentiated the cytotoxic activity of TRAIL in RKO and HT29 cells and that of L-OHP in HT29 cells. Similarly, overexpression of IkappaBalpha mutant (IkappaBalphaM) or treatment with the IKK inhibitor, BMS-345541, also sensitized these cells to TRAIL and L-OHP. Importantly, 2 h of quinacrine pretreatment resulted in decreased expression of c-FLIP and Mcl-1, which were determined to be transcriptional targets of NF-kappaB. Extended exposure for 24 h to quinacrine did not further sensitize these cells to TRAIL- or L-OHP-induced cell death; however, exposure caused the down-regulation of additional NF-kappaB-dependent survival factors. Short hairpin RNA-mediated knockdown of c-FLIP or Mcl-1 significantly sensitized these cells to TRAIL and L-OHP. Taken together, data demonstrate that NF-kappaB is constitutively active in colon cancer cell lines and NF-kappaB, and its downstream targets may constitute an important target for the development of therapeutic approaches against this disease.

Myc regulates aggresome formation, the induction of Noxa, and apoptosis in response to the combination of bortezomib and SAHA.

The histone deacetylase inhibitor SAHA enhances cell death stimulated by the proteasome inhibitor bortezomib (BZ) by disrupting BZ-induced aggresome formation. Here we report that Myc regulates the sensitivity of multiple myeloma (MM) cells to BZ + SAHA-induced cell death. In MM cells, Myc expression directly correlated with intracellular ER content, protein synthesis rates, the percentage of aggresome-positive cells, and the sensitivity to BZ + SAHA-induced cell death. Accordingly, Myc knockdown markedly reduced the sensitivity of MM cells to BZ + SAHA-mediated apoptosis. Furthermore, activation of Myc was sufficient to provoke aggresome formation and thus sensitivity to BZ + SAHA, and these responses required de novo protein synthesis. BZ + SAHA-mediated stimulation of apoptosis includes the induction of the proapoptotic BH3-only protein Noxa as well as endoplasmic reticular stress, a disruption of calcium homeostasis, and activation of capase-4. Finally, knockdown studies demonstrated that both caspase-4 and Noxa play significant roles in Myc-driven sensitivity to BZ + SAHA-induced apoptosis. Collectively, our results establish a mechanistic link between Myc activity, regulation of protein synthesis, increases in HDAC6 expression and aggresome formation, induction of Noxa, and sensitivity to BZ + SAHA-induced apoptosis. These data suggest that MM patients with elevated Myc activity may be particularly sensitive to the BZ + SAHA combination.

ATP modulates PTEN subcellular localization in multiple cancer cell lines.

The tumour suppressor gene PTEN plays an important somatic role in both hereditary and sporadic breast carcinogenesis. While the role of PTEN's lipid phosphatase activity, as a negative regulator of the cytoplasmic phosphatidylinositol-3-kinase/Akt pathway is well known, it is now well established that PTEN exists and functions in the nucleus. Multiple mechanisms of regulating PTEN's subcellular localization have been reported. However none are ubiquitous across multiple cancer cell lines and tissue types. We show here that adenosine triphosphate (ATP) regulates PTEN subcellular localization in a variety of different cancer cell lines, including those derived from breast, colon and thyroid carcinomas. Cells deficient in ATP show an increased level of nuclear PTEN protein. This increase in PTEN is reversed when cells are supplemented with ATP, ADP or AMP. In contrast, the addition of the non-hydrolyzable analogue ATPgammaS, did not reverse nuclear PTEN protein levels in all the cell types tested. To our knowledge, this is the first report that describes a regulation of PTEN subcellular localization that is not specific to one cell line or tissue type, but appears to be common across a variety of cell lineages.

The novel polyamine analogue CGC-11093 enhances the antimyeloma activity of bortezomib.

Multiple myeloma (MM) is an incurable plasma cell malignancy. The recent successes of the proteasome inhibitor bortezomib in MM therapy have prompted investigations of its efficacy in combination with other anticancer agents. Polyamines play important roles in regulating tumor cell proliferation and angiogenesis and represent an important therapeutic target. CGC-11093 is a novel polyamine analogue that has completed a phase I clinical trial for the treatment of cancer. Here, we report that CGC-11093 selectively augments the in vitro and in vivo antimyeloma activity of bortezomib. Specifically, the combination of CGC-11093 and bortezomib compromised MM viability and clonogenic survival, and increased drug-induced apoptosis over that achieved by either single agent. Xenografts of MM tumors treated with this combination had marked increases in phospho-c-Jun-NH(2)-kinase (JNK)-positive cells and apoptosis, and corresponding reductions in tumor burden, tumor vasculature, and the expression of proliferating cell nuclear antigen and the proangiogenic cytokine vascular endothelial growth factor. Furthermore, inhibition of JNK with a pharmacologic inhibitor or by selective knockdown blunted the efficacy of CGC-11093 and bortezomib. Therefore, CGC-11093 enhances the anticancer activity of bortezomib by augmenting JNK-mediated apoptosis and blocking angiogenesis. These findings support the study of the use of the combination of bortezomib and CGC-11093 in MM patients that fail to respond to frontline therapy.

Histone deacetylase inhibitors enhance lexatumumab-induced apoptosis via a p21Cip1-dependent decrease in survivin levels.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in malignant cells by binding to the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Several agents that therapeutically exploit this phenomenon are being developed. We investigated the anticancer activity of two novel, highly specific agonistic monoclonal antibodies to TRAIL-R1 (mapatumumab, HGS-ETR1) and TRAIL-R2 (lexatumumab, HGS-ETR2) in colon cancer cell lines. Our analyses revealed that colon cancer cells display significantly higher surface expressions of TRAIL-R2 than TRAIL-R1, and are more sensitive to lexatumumab-induced apoptosis. The proapoptotic effects of lexatumumab in TRAIL-resistant HCT8 and HT29 cells were dramatically augmented by the histone deacetylase inhibitors trichostatin A or suberoylanilide hydroxamic acid. The presence of p21, but not p53, was critical for the synergy between lexatumumab and histone deacetylase inhibitors. The absence of p21 did not interfere with the formation of the death-inducing signaling complex by lexatumumab, suggesting the involvement of other apoptotic and/or cell cycle regulators. Indeed, treatment with suberoylanilide hydroxamic acid greatly reduced the expression of the inhibitor of apoptosis protein survivin and cdc2 activity in HCT116 p21(+/+) cells but not in the HCT116 p21(-/-) cells. Inhibition of cdc2 activity with flavopiridol decreased survivin expression and sensitized the p21-deficient cells to lexatumumab-induced apoptosis. Similarly, small interfering RNA-mediated knockdown of survivin also enhanced lexatumumab-mediated cell death. Therefore, survivin expression plays a key role in lexatumumab resistance, and reducing survivin expression by inhibiting cdc2 activity is a promising strategy to enhance the anticancer activity of lexatumumab.

Targeting autophagy augments the anticancer activity of the histone deacetylase inhibitor SAHA to overcome Bcr-Abl-mediated drug resistance.

Novel therapeutic strategies are needed to address the emerging problem of imatinib resistance. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) is being evaluated for imatinib-resistant chronic myelogenous leukemia (CML) and has multiple cellular effects, including the induction of autophagy and apoptosis. Considering that autophagy may promote cancer cell survival, we hypothesized that disrupting autophagy would augment the anticancer activity of SAHA. Here we report that drugs that disrupt the autophagy pathway dramatically augment the antineoplastic effects of SAHA in CML cell lines and primary CML cells expressing wild-type and imatinib-resistant mutant forms of Bcr-Abl, including T315I. This regimen has selectivity for malignant cells and its efficacy was not diminished by impairing p53 function, another contributing factor in imatinib resistance. Disrupting autophagy by chloroquine treatment enhances SAHA-induced superoxide generation, triggers relocalization and marked increases in the lysosomal protease cathepsin D, and reduces the expression of the cathepsin-D substrate thioredoxin. Finally, knockdown of cathepsin D diminishes the potency of this combination, demonstrating its role as a mediator of this therapeutic response. Our data suggest that, when combined with HDAC inhibitors, agents that disrupt autophagy are a promising new strategy to treat imatinib-refractory patients who fail conventional therapy.

Sphingosine-induced apoptosis in rhabdomyosarcoma cell lines is dependent on pre-mitochondrial Bax activation and post-mitochondrial caspases.

Sphingolipids is the collective term ascribed to components of the sphingomyelin cycle. Modulation of the cellular levels of individual sphingolipids can induce a diverse range of cellular responses including apoptosis, proliferation, and cell cycle arrest. We present data showing that rhabdomyosarcoma cell lines, independent of lineage (alveolar rhabdomyosarcoma and embryonal rhabdomyosarcoma), are particularly sensitive to the induction of apoptosis as a result of an elevation in the cellular levels of sphingosine (D-erythro-sphingosine). Sphingosine-mediated apoptosis does not require its metabolism to the related proapoptotic molecule ceramide and is stereospecific because exposure of the rhabdomyosarcoma cell line RD to the L-erythro and DL-threo isoforms of sphingosine did not induce apoptosis. Importantly, for efficient induction of apoptosis, sphingosine required Bax activation and consequential translocation to the mitochondria. This resulted in selective mitochondrial release of cytochrome c and Smac/Diablo but not other mitochondrial related factors (apoptosis-inducing factor, endonuclease G, and HtrA2/Omi). Using small interfering RNA, reduced Bax expression conferred the impaired release of mitochondrial cytochrome c to the cytoplasm following sphingosine exposure, inhibiting the induction of apoptosis. Furthermore, dissipation of the inner mitochondrial membrane potential and enhanced production of reactive oxygen species were not observed. Bax activation and cytochrome c release were independent of caspases; however, caspase-3 and caspase-9 activity distal to the mitochondria was essential for the execution of apoptosis.

Proteasome inhibitors sensitize colon carcinoma cells to TRAIL-induced apoptosis via enhanced release of Smac/DIABLO from the mitochondria.

The synergistic interaction between proteasome inhibitors and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising approach to induce cell death in tumor cells. However, the molecular and biochemical mechanisms of this synergism have been proven to be cell type specific. We therefore focused our investigation on TRAIL-resistant colon carcinoma cells in this study. DNA fragmentation, mitochondrial membrane depolarization and increased caspase-3-like enzyme activity was exclusively induced only by combined treatment with proteasome inhibitors (epoxomicin, MG132, bortezomib/PS-341) and TRAIL. The expression level of anti-apoptotic proteins (XIAP, survivin, Bcl-2, Bcl-XL), regulated by NF-kappaB transcription factor, was not effected by any of these treatments. TRAIL alone induced only partial activation of caspase-3 (p20), while the combination of TRAIL and proteasome inhibition led to the full proteolytic activation of caspase-3 (p17). Only the combination treatment induced marked membrane depolarization and the release of cytochrome c, HtrA2/Omi and Smac/DIABLO. Apoptosis-inducing factor (AIF) was not released in any of these conditions. These results are consistent with a model where the full activation of caspase-3 by caspase-8 is dependent on the release of Smac/DIABLO in response to the combined treatment. This molecular mechanism, independent of the inhibition NF-kappaB activity, may provide rationale for the combination treatment of colon carcinomas with proteasome inhibitors and recombinant TRAIL or agonistic antibody of TRAIL receptors.

Cyclooxygenase-2 inhibition sensitizes human colon carcinoma cells to TRAIL-induced apoptosis through clustering of DR5 and concentrating death-inducing signaling complex components into ceramide-enriched caveolae.

Cyclooxygenase-2 (COX-2) is up-regulated in human colon carcinomas, and its inhibition is associated with a reduction in tumorigenesis and a promotion of apoptosis. However, the mechanisms responsible for the antitumor effects of COX-2 inhibitors and how COX-2 modulates apoptotic signaling have not been clearly defined. We have shown that COX-2 inhibition sensitizes human colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by inducing clustering of the TRAIL receptor DR5 at the cell surface and the redistribution of the death-inducing signaling complex components (DR5, FADD, and procaspase-8) into cholesterol-rich and ceramide-rich domains known as caveolae. This process requires the accumulation of arachidonic acid and sequential activation of acid sphingomyelinase for the generation of ceramide within the plasma membrane outer leaflet. The current study highlights a novel mechanism to circumvent colorectal carcinoma cell resistance to TRAIL-mediated apoptosis using COX-2 inhibitors to manipulate the lipid metabolism within the plasma membrane.