F4/80+Ly6Chigh Macrophages Lead to Cell Plasticity and Cancer Initiation in Colitis.

BACKGROUND & AIMS: Colorectal cancer is a leading cause of cancer death, and a major risk factor is chronic inflammation. Despite the link between colitis and cancer, the mechanism by which inflammation leads to colorectal cancer is not well understood. METHODS: To investigate whether different forms of inflammation pose the same risk of cancer, we compared several murine models of colitis (dextran sodium sulfate [DSS], 2,4,6-trinitrobenzene sulfonic acid, 4-ethoxylmethylene-2-phenyloxazol-5-one, Citrobacter rodentium, Fusobacterium nucleatum, and doxorubicin) with respect to their ability to lead to colonic tumorigenesis. We attempted to correlate the severity of colitis and inflammatory profile with the risk of tumorigenesis in both azoxymethane-dependent and Dclk1/APCfl/fl murine models of colitis-associated cancer. RESULTS: DSS colitis reproducibly led to colonic tumors in both mouse models of colitis-associated cancer. In contrast, all other forms of colitis did not lead to cancer. When compared with the colitis not associated with tumorigenesis, DSS colitis was characterized by significantly increased CD11b+F4/80+Ly6Chigh macrophages and CD11b+Ly6G+ neutrophils. Interestingly, depletion of the CD11b+F4/80+Ly6Chigh macrophages inhibited tumorigenesis, whereas depletion of CD11b+Ly6G+ neutrophils had no effect on tumorigenesis. Furthermore, the macrophage-derived cytokines interleukin-1ß, tumor necrosis factor-α, and interleukin-6 were significantly increased in DSS colitis and promoted stemness of Dclk1+ tuft cells that serve as the cellular origin of cancer. CONCLUSIONS: We have identified CD11b+F4/80+Ly6Chigh macrophages as key mediators of cancer initiation in colitis-associated cancer. Development of new therapies that target these cells may provide an effective preventative strategy for colitis-associated cancer.

Intestinal Stem Cell Marker ASCL2 is a Novel Prognostic Predictor in Esophageal Adenocarcinoma.

Purpose Intestinal stem cell markers play a significant role in esophageal adenocarcinoma carcinogenesis via Barrett's esophagus; however, its utility as a prognostic biomarker has not been established. Methods We analyzed the immunohistochemical expression of intestinal stem cell markers, ASCL2 and LGR5, using whole slides (35 cases) and tissue microarray (TMA; 64 cases). On TMA slides, adjacent normal squamous epithelium, metaplastic glandular epithelium (Barrett's esophagus), and dysplastic glandular epithelium were inserted when applicable. Two pathologists semi-quantitatively scored stained slides independently, and the results were correlated with clinicopathologic factors and outcomes. Results In whole slides, 51% and 57% expressed high ASCL2 and high LGR5; in TMA, 69% and 88% expressed high ASCL2 and high LGR5, respectively. In TMA, high ASCL2 and low LGR5 expression significantly correlated to a higher number of involved lymph nodes (p=0.027 and p=0.0039), and LGR5 expression significantly correlated to the pathological stage (p=0.0032). Kaplan-Meier analysis showed a negative impact of high ASCL2 expression on overall survival (OS; WS p=0.0168, TMA p=0.0276) as well as progression-free survival (PFS; WS p=0.000638, TMA p=0.0466) but not LGR5. Multivariate Cox regression analysis revealed that ASCL2 expression is an independent prognostic factor for esophageal adenocarcinoma (OS; WS p=0.25, TMA p=0.011. PFS; WS p=0.012, TMA p=0.038). Analysis of the TCGA dataset showed that ASCL2 mRNA levels were correlated to nodal status but not overall survival. Conclusion High expression of the intestinal stem cell marker ASCL2 may predict unfavorable outcomes in surgically resected esophageal adenocarcinoma.

scSNV: accurate dscRNA-seq SNV co-expression analysis using duplicate tag collapsing.

Identifying single nucleotide variants has become common practice for droplet-based single-cell RNA-seq experiments; however, presently, a pipeline does not exist to maximize variant calling accuracy. Furthermore, molecular duplicates generated in these experiments have not been utilized to optimally detect variant co-expression. Herein, we introduce scSNV designed from the ground up to "collapse" molecular duplicates and accurately identify variants and their co-expression. We demonstrate that scSNV is fast, with a reduced false-positive variant call rate, and enables the co-detection of genetic variants and A>G RNA edits across twenty-two samples.

Towards personalized induction therapy for esophageal adenocarcinoma: organoids derived from endoscopic biopsy recapitulate the pre-treatment tumor.

Esophageal adenocarcinoma has few known recurrent mutations and therefore robust, reliable and reproducible patient-specific models are needed for personalized treatment. Patient-derived organoid culture is a strategy that may allow for the personalized study of esophageal adenocarcinoma and the development of personalized induction therapy. We therefore developed a protocol to establish EAC organoids from endoscopic biopsies of naïve esophageal adenocarcinomas. Histologic characterization and molecular characterization of organoids by whole exome sequencing demonstrated recapitulation of the tumors' histology and genomic (~ 60% SNV overlap) characteristics. Drug testing using clinically appropriate chemotherapeutics and targeted therapeutics showed an overlap between the patient's tumor response and the corresponding organoids' response. Furthermore, we identified Barrett's esophagus epithelium as a potential source of organoid culture contamination. In conclusion, organoids can be robustly cultured from endoscopic biopsies of esophageal adenocarcinoma and recapitulate the originating tumor. This model demonstrates promise as a tool to better personalize therapy for esophageal adenocarcinoma patients.

Author Correction: Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.

A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.

miR-145 expression enhances integrin expression in SK-GT-4 cell line by down-regulating c-Myc expression.

Adenocarcinoma of the esophagus is increasing in frequency and is the 6th most common cause of cancer death in North America. In adenocarcinoma cell lines, we have previously demonstrated that expression of miR-145, leads to enhanced invasion, resistance to anoikis and better attachment to fibronectin in esophageal adenocarcinoma. In contrast, expression of miR-145 acts as a tumor suppressor in squamous cell carcinoma. The molecular mechanisms responsible for the oncogenic effects of miR-145 were investigated. In this report, we demonstrate that we can partially recreate the miR-145 effects in EAC by knock down of the expression of c-Myc, which is one of the targets of miR-145. Knocking down of c-Myc expression resulted in upregulation of integrin subunits α5 and ß3. Finally, we demonstrated that integrin α5 expression correlates to fibronectin attachment potential whereas integrin ß3 expression correlates with resistance to anoikis and invasion potential. Finally, we demonstrate that expression of miR-145 in esophageal adenocarcinoma cell line (SK-GT-4) enhances tumor growth and metastasis in a NOD/SCID xenograft model. Overall, the oncogenic potential of miR-145 in EAC appears to be mediated by downregulation of c-Myc leading to the expression of integrins subunits α5 and ß3.

Partial Restoration of CFTR Function in cftr-Null Mice following Targeted Cell Replacement Therapy.

Cystic fibrosis (CF) is a fatal recessive genetic disorder caused by a mutation in the gene encoding CF transmembrane conductance regulator (CFTR) protein. Alteration in CFTR leads to thick airway mucus and bacterial infection. Cell therapy has been proposed for CFTR restoration, but efficacy has been limited by low engraftment levels. In our previous studies, we have shown that using a pre-conditioning regimen in combination with optimization of cell number and time of delivery, we could obtain greater bone marrow cell (BMC) retention in the lung. Here, we found that optimized delivery of wild-type (WT) BMC contributed to apical CFTR expression in airway epithelium and restoration of select ceramide species and fatty acids in CFTR-/- mice. Importantly, WT BMC delivery delayed Pseudomonas aeruginosa lung infection and increased survival of CFTR-/- recipients. Only WT BMCs had a beneficial effect beyond 6 months, suggesting a dual mechanism of BMC benefit: a non-specific effect early after cell delivery, possibly due to the recruitment of macrophages and neutrophils, and a late beneficial effect dependent on long-term CFTR expression. Taken together, our results suggest that BMC can improve overall lung function and may have potential therapeutic benefit for the treatment of CF.

Stimulus-dependent dissociation between XB130 and Tks5 scaffold proteins promotes airway epithelial cell migration.

Repair of airway epithelium after injury requires migration of neighboring epithelial cells to injured areas. However, the molecular mechanisms regulating airway epithelial cell migration is not well defined. We have previously shown that XB130, a scaffold protein, is required for airway epithelial repair and regeneration in vivo, and interaction between XB130 and another scaffold protein, Tks5, regulates cell proliferation and survival in human bronchial epithelial cells. The objective of the present study was to determine the role of XB130 and Tks5 interaction in airway epithelial cell migration. Interestingly, we found that XB130 only promotes lateral cell migration, whereas, Tks5 promotes cell migration/invasion via proteolysis of extracellular matrix. Upon stimulation with EGF, PKC activator phorbol 12, 13-dibutyrate or a nicotinic acetylcholine receptor ligand, XB130 and Tks5 translocated to the cell membrane in a stimulus-dependent manner. The translocation and distribution of XB130 is similar to lamellipodial marker, WAVE2; whereas Tks5 is similar to podosome marker, N-WASP. Over-expression of XB130 or Tks5 alone enhances cell migration, whereas co-expression of both XB130 and Tks5 inhibits cell migration processes and signaling. Furthermore, XB130 interacts with Rac1 whereas Tks5 interacts with Cdc42 to promote Rho GTPase activity. Our results suggest that dissociation between XB130 and Tks5 may facilitate lateral cell migration via XB130/Rac1, and vertical cell migration via Tks5/Cdc42. These molecular mechanisms will help our understanding of airway epithelial repair and regeneration.

Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.

New biological tools are required to understand the functional significance of genetic events revealed by whole genome sequencing (WGS) studies in oesophageal adenocarcinoma (OAC). The MFD-1 cell line was isolated from a 55-year-old male with OAC without recombinant-DNA transformation. Somatic genetic variations from MFD-1, tumour, normal oesophagus, and leucocytes were analysed with SNP6. WGS was performed in tumour and leucocytes. RNAseq was performed in MFD-1, and two classic OAC cell lines FLO1 and OE33. Transposase-accessible chromatin sequencing (ATAC-seq) was performed in MFD-1, OE33, and non-neoplastic HET1A cells. Functional studies were performed. MFD-1 had a high SNP genotype concordance with matched germline/tumour. Parental tumour and MFD-1 carried four somatically acquired mutations in three recurrent mutated genes in OAC: TP53, ABCB1 and SEMA5A, not present in FLO-1 or OE33. MFD-1 displayed high expression of epithelial and glandular markers and a unique fingerprint of open chromatin. MFD-1 was tumorigenic in SCID mouse and proliferative and invasive in 3D cultures. The clinical utility of whole genome sequencing projects will be delivered using accurate model systems to develop molecular-phenotype therapeutics. We have described the first such system to arise from the oesophageal International Cancer Genome Consortium project.

Cancer-associated fibroblasts predict poor outcome and promote periostin-dependent invasion in oesophageal adenocarcinoma.

Interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an important role in tumour development and progression. In this study we investigated the functional role of CAFs in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of 183 EAC patients for CAF markers related to disease mortality. We characterized CAFs and normal oesophageal fibroblasts (NOFs) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3D organotypic culture and xenograft models were used to examine the effects on EAC cell function and to dissect molecular mechanisms regulating invasion. Most EACs (93%) contained CAFs with a myofibroblastic (α-SMA-positive) phenotype, which correlated significantly with poor survival [p = 0.016; HR 7. 1 (1.7-29.4)]. Primary CAFs isolated from EACs have a contractile, myofibroblastic phenotype and promote EAC cell invasion in vitro (Transwell assays, p ≤ 0.05; organotypic culture, p < 0.001) and in vivo (p ≤ 0.05). In vitro, this pro-invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAFs and acts as a ligand for EAC cell integrins αvß3 and αvß5, promoting activation of the PI3kinase-Akt pathway. In patient samples, periostin expression at the tumour cell-stromal interface correlates with poor overall and disease-free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF-secreted periostin and EAC-expressed integrins results in PI3 kinase-Akt activation and increased tumour cell invasion. Most EACs contain a myofibroblastic CAF-rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patients.

MiR-145 expression accelerates esophageal adenocarcinoma progression by enhancing cell invasion and anoikis resistance.

BACKGROUND: Carcinoma of the esophagus has a high case fatality ratio and is now the 6th most common cause of cancer deaths in the world. We previously conducted a study to profile the expression of miRNAs in esophageal adenocarcinoma (EAC) pre and post induction therapy. Of the miRNAs differentially expressed post induction chemoradiation, miR-145, a known tumor suppressor miRNA, was upregulated 8-fold following induction therapy, however, its expression was associated with shorter disease-free survival. This unexpected result was explored in this current study. METHODS: In order to study the role of miR-145 in EAC, miRNA-145 was overexpressed in 3 EAC cell lines (OE33, FLO-1, SK-GT-4) and one ESCC cell line (KYSE-410). After validation of the expression of miR-145, hallmarks of cancer such as cell proliferation, resistance to chemotherapy drugs or anoikis, and cell invasion were analyzed. RESULTS: There were no differences in cell proliferation and 5 FU resistance between miR145 cell lines and the control cell lines. miR-145 expression also had no effect on cisplatin resistance in two of three cell lines (OE33 and FLO-1), but miR-145 appeared to protect SK-GT-4 cells against cisplatin treatment. However, there was a significant difference in cell invasion, cell adhesion and resistance to anoikis. All three EAC miR-145 cell lines invaded more than their respective controls. Similarly, OE33 and SK-GT-4 miR-145 cell lines were able to survive longer in a suspension state. DISCUSSION: While expression of miR-145 in ESCC stopped proliferation and invasion, expression of miR-145 in EAC cells enhanced invasion and anoikis resistance. Although more work is required to understand how miR-145 conveys these effects, expression of miR-145 appears to promote EAC progression by enhancing invasion and protection against anoikis, which could in turn facilitate distant metastasis.

A comparison of primary oesophageal squamous epithelial cells with HET-1A in organotypic culture.

BACKGROUND INFORMATION: Carcinoma of the oesophagus is the sixth leading cause of cancer death in the western world and is associated with a 5-year survival of less than 15%. Recent evidence suggests that stromal-epithelial interactions are fundamental in carcinogenesis. The advent of co-culture techniques permits the investigation of cross-talk between the stroma and epithelium in a physiological setting. We have characterized a histologically representative oesophageal organotypic model and have used it to compare the most commonly used squamous oesophageal cell line, HET-1A, with primary oesophageal squamous cells for use in studies of the oesophageal epithelium in vitro. RESULTS: When grown in an organotypic culture with normal fibroblasts, the oesophageal carcinoma cell lines OE21 (squamous) and OE19 (adenocarcinoma) morphologically resembled the tumour of origin with evidence of stromal invasion and mucus production, respectively. However, HET-1A cells, which were derived from normal squamous oesophageal cells, appeared dysplastic and failed to display evidence of squamous differentiation. By comparison with primary oesophageal epithelial cells, the HET-1A cells were highly proliferative and did not express the epithelial markers E-cadherin or CK5/6 (casein kinase 5/6), or the stratified epithelial marker ΔNp63, but did express the mesenchymal markers vimentin and N-cadherin. CONCLUSION: Studies of epithelial carcinogenesis will benefit from culture systems which allow manipulation of the stromal and epithelial layers independently. We have developed an organotypic culture using primary oesophageal squamous cells and fibroblasts in which a stratified epithelium with a proliferative basal layer that stains strongly for ΔNp63 develops. This model will be suitable for the study of the molecular events in the development of Barrett's oesophagus. The most commonly used normal oesophageal squamous cell line, HET-1A, does not have the characteristics of normal oesophageal squamous cells and should not be used in models of the normal oesophageal epithelium. Until more representative cell lines are available, future studies in oesophageal cancer will be reliant on the availability and manipulation of primary tissue.

Hypoxia-induced downregulation of autophagy mediator Beclin 1 reduces the susceptibility of malignant intestinal epithelial cells to hypoxia-dependent apoptosis.

Disruption of tumor blood supply causes tumor hypoxia. Hypoxia can induce cell death, but cancer cells that remain viable in the absence of oxygen often possess an increased survival potential, and tumors formed by these cells tend to grow particularly aggressively. Thus, developing approaches aimed at increasing the susceptibility of malignant cells to hypoxia-induced death represents a potentially important avenue for cancer treatment. Molecular mechanisms that control the survival of cancer cells under hypoxia are not well understood. In an effort to understand them we found that hypoxia downregulates Beclin 1, a mediator of autophagy, in malignant intestinal epithelial cells. The reversal of this downregulation promoted autophagosome accumulation, enhanced the activation of a pro-apoptotic protease caspase-9 and subsequent caspase-9-dependent activation of two other pro-apoptotic proteases caspases 3 and 7 in these cells. Furthermore, the reversal of hypoxia-induced downregulation of Beclin 1-stimulated caspase-9-dependent apoptosis of the indicated cells under hypoxia. Interestingly, we found that Beclin 1-dependent caspase-9 activation in hypoxic cells was not associated with an increased release of cytochrome c from the mitochondria to the cytoplasm (such release represents a frequently occurring mechanism for caspase-9 activation). We also observed that Beclin 1-dependent apoptosis of hypoxic malignant cells was independent of FADD, a mediator of death receptor signaling. We conclude that hypoxia triggers a feedback mechanism that delays apoptosis of oxygen-deprived malignant intestinal epithelial cells and is driven by hypoxia-induced Beclin 1 downregulation. Thus, approaches aimed at the disruption of this mechanism can be expected to enhance the susceptibility of such cells to hypoxia-induced apoptosis.

Palmitoylation of the TRAIL receptor DR4 confers an efficient TRAIL-induced cell death signalling.

S-palmitoylation is a lipid modification that regulates membrane-protein association and influences protein trafficking, stability or aggregation, thus playing an important role in protein signalling. We previously demonstrated that the palmitoylation of Fas, one of the DD (death domain)-containing members of the TNFR [TNF (tumour necrosis factor) receptor] superfamily, is essential for the redistribution of this receptor into lipid rafts, an obligatory step for the death signal transmission. Here we investigate the requirement of protein palmitoylation in the activities of other DD-containing death receptors. We show that DR4 is palmitoylated, whereas DR5 and TNFR1 are not. Furthermore, DR4 palmitoylation is required for its raft localization and its ability to oligomerize, two essential features in TRAIL (TNF-related apoptosis-inducing ligand)-induced death signal transmission.

Acquisition of anoikis resistance promotes the emergence of oncogenic K-ras mutations in colorectal cancer cells and stimulates their tumorigenicity in vivo.

Detachment from the extracellular matrix causes apoptosis of normal epithelial cells--a phenomenon called anoikis. K-ras oncogene, an established anoikis inhibitor, often occurs in colorectal carcinoma (CRC). In addition to blocking anoikis-inducing mechanisms, oncogenic K-ras can cause anoikis-unrelated changes in CRC cells, such as induction of events promoting their deregulated mitogenesis, ability to trigger angiogenesis, and so on. Thus, whether ras-induced anoikis resistance of CRC cells is essential for their ability to form tumors in vivo or represents a mere epiphenomenon is unclear. We found that when poorly tumorigenic, oncogenic, K-ras-negative, anoikis-susceptible human CRC cells were cultured under anoikis-inducing conditions in vitro, they spontaneously gave rise to an anoikis-resistant cell population harboring de novo oncogenic K-ras mutations and manifesting dramatically increased tumorigenicity. We further observed that a variant of the same oncogenic K-ras-negative anoikis-susceptible cells selected for increased tumorigenicity acquired de novo oncogenic K-ras mutations and manifested increased anoikis resistance. Unlike the case with anoikis, oncogenic K-ras did not rescue CRC cells from death caused by hypoxia or anticancer agents. Taken collectively, our results support the notion that ras-induced anoikis resistance of CRC cells is essential for their ability to form tumors in vivo and thus represents a potential therapeutic target.

Oncogenic Ras inhibits anoikis of intestinal epithelial cells by preventing the release of a mitochondrial pro-apoptotic protein Omi/HtrA2 into the cytoplasm.

Resistance of cancer cells to anoikis, apoptosis induced by cell detachment from the extracellular matrix, is thought to represent a critical feature of the malignant phenotype. Mechanisms that control anoikis of normal and cancer cells are understood only in part. Previously we found that anoikis of non-malignant intestinal epithelial cells is driven by detachment-induced down-regulation of Bcl-X(L), a protein that blocks apoptosis through preventing the release of death-promoting factors from the mitochondria. Mitochondrial proteins the release of which causes anoikis are presently unknown. Similar to what was previously observed by others for keratinocytes and fibroblasts, we show here that anoikis of intestinal epithelial cells does not involve caspase-9, a target of a mitochondrial protein cytochrome c. Furthermore, Smac/Diablo, another mitochondrial pro-apoptotic factor, does not appear to play a role in detachment-dependent apoptosis of these cells either. Instead, anoikis of intestinal epithelial cells is triggered by the release of a mitochondrial protein Omi/HtrA2, an event driven by detachment-induced down-regulation of Bcl-X(L). Moreover, we established that oncogenic ras inhibits anoikis by preventing the release of Omi/HtrA2. This effect of ras required ras-induced down-regulation of a pro-apoptotic protein Bak and could be blocked by an inhibitor of phosphoinositide 3-kinase, a target of Ras that was previously implicated by us in the down-regulation of Bak and blockade of anoikis. We conclude that Omi/HtrA2 is an inducer of anoikis and an important regulator of ras-induced transformation.

Sodium salicylate promotes neutrophil apoptosis by stimulating caspase-dependent turnover of Mcl-1.

Mcl-1 is an antiapoptotic member of the Bcl-2 family of proteins that plays a central role in cell survival of neutrophils and other cells. The protein is unusual among family members in that it has a very short half-life of 2-3 h. In this report, we show that sodium salicylate (at 10 mM) greatly enhances the rate at which neutrophils undergo apoptosis and, in parallel, greatly accelerates the turnover rate of Mcl-1, decreasing its half-life to only 90 min. Whereas constitutive and GM-CSF-modified Mcl-1 turnover is regulated by the proteasome, the accelerated sodium salicylate-induced Mcl-1 turnover is mediated largely via caspases. Sodium salicylate resulted in rapid activation of caspase-3, -8, -9, and -10, and salicylate-accelerated Mcl-1 turnover was partly blocked by caspase inhibitors. Sodium salicylate also induced dramatic changes in the activities of members of the MAPK family implicated in Mcl-1 turnover and apoptosis. For example, sodium salicylate blocked GM-CSF-stimulated Erk and Akt activation, but resulted in rapid and sustained activation of p38-MAPK, an event mimicked by okadaic acid that also accelerates Mcl-1 turnover and neutrophil apoptosis. These data thus shed important new insights into the dynamic and highly regulated control of neutrophil apoptosis that is effected by modification in the rate of Mcl-1 turnover.

ras Oncogene triggers up-regulation of cIAP2 and XIAP in intestinal epithelial cells: epidermal growth factor receptor-dependent and -independent mechanisms of ras-induced transformation.

Detachment of normal epithelial cells from the extracellular matrix (ECM) triggers apoptosis, a phenomenon called anoikis. Conversely, carcinomas (cancers of epithelial origin) represent three-dimensional disorganized multicellular masses in which cells are deprived of adhesion to the ECM but remain viable. Resistance of cancer cells to anoikis is thought to be critical for tumor progression. However, the knowledge about molecular mechanisms of this type of resistance remains limited. Herein we report that ras oncogene, an established inhibitor of anoikis, triggers a significant upregulation of anti-apoptotic proteins cIAP2 and XIAP in intestinal epithelial cells. We also observed that the effect of ras on cIAP2 requires ras-induced autocrine production of transforming growth factor alpha (TGF-alpha), a ligand for epidermal growth factor receptor, whereas ras-triggered up-regulation of XIAP is TGF-alpha-independent. Moreover, overexpression of either cIAP2 or XIAP in nonmalignant intestinal epithelial cell was found to block anoikis. In addition, an established IAP antagonist Smac or Smac-derived cell-permeable peptide suppressed ras-induced anoikis resistance and subsequent anchorage-independent growth of ras-transformed cells. We conclude that ras-induced overexpression of cIAP2 and XIAP significantly contributes to the ability of ras-transformed intestinal epithelial cells to survive in the absence of adhesion to the ECM and grow in a three-dimensional manner.

Granulocyte macrophage colony-stimulating factor signaling and proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1.

Human neutrophils normally have a very short half-life and die by apoptosis. Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) can delay this apoptosis via increases in the cellular levels of Mcl-1, an anti-apoptotic protein of the Bcl-2 family with a rapid turnover rate. Here we have shown that inhibition of the proteasome (a) decreases the rate of Mcl-1 turnover within neutrophils and (b) significantly delays apoptosis. This led us to determine whether GM-CSF could enhance neutrophil survival by altering the rate of Mcl-1 turnover. Addition of GM-CSF to neutrophils enhanced Mcl-1 stability and delayed apoptosis by signaling pathways requiring PI3K/Akt and p44/42 Erk/Mek, because inhibitors of these pathways completely abrogated the GM-CSF-mediated effect on both Mcl-1 stability and apoptosis delay. Conversely, induction of Mcl-1 hyperphosphorylation by the phosphatase inhibitor, okadaic acid, significantly accelerated both Mcl-1 turnover and apoptosis. Neither the calpain inhibitor, carbobenzoxy-valinyl-phenylalaninal, nor the pan caspase inhibitor, benzyloxycarbonyl-VAD-fluoromethylketone, had any effect on Mcl-1 stability under these conditions. These observations indicate that profound changes in the rate of neutrophil apoptosis following cytokine signaling occur via dynamic changes in the rate of Mcl-1 turnover via the proteasome.