Defective quorum sensing of acute lymphoblastic leukemic cells: evidence of collective behavior of leukemic populations as semi-autonomous aberrant ecosystems.

Quorum sensing (QS) is a generic term used to describe cell-cell communication and collective decision making by bacterial and social insects to regulate the expression of specific genes in controlling cell density and other properties of the populations in response to nutrient supply or changes in the environment. QS mechanisms also have a role in higher organisms in maintaining homeostasis, regulation of the immune system and collective behavior of cancer cell populations. In the present study, we used a p190(BCR-ABL) driven pre-B acute lymphoblastic leukemia (ALL3) cell line derived from the pleural fluid of a terminally ill patient with ALL to test the QS hypothesis in leukemia. ALL3 cells don't grow at low density (LD) in liquid media but grow progressively faster at increasingly high cell densities (HD) in contrast to other established leukemic cell lines that grow well at very low starting cell densities. The ALL3 cells at LD are poised to grow but shortly die without additional stimulation. Supernates of ALL3 cells (HDSN) and some other primary cells grown at HD stimulate the growth of the LD ALL3 cells without which they won't survive. To get further insight into the activation processes we performed microarray analysis of the LD ALL3 cells after stimulation with ALL3 HDSN at days 1, 3, and 6. This screen identified several candidate genes, and we linked them to signaling networks and their functions. We observed that genes involved in lipid, cholesterol, fatty acid metabolism, and B cell activation are most up- or down-regulated upon stimulation of the LD ALL3 cells using HDSN. We also discuss other pathways that are differentially expressed upon stimulation of the LD ALL3 cells. Our findings suggest that the Ph+ ALL population achieves dominance by functioning as a collective aberrant ecosystem subject to defective quorum-sensing regulatory mechanisms.

Automated mass spectrometry-based functional assay for the routine analysis of the secretome.

The secretome represents the set of proteins secreted into the extracellular space of cells. These proteins have been shown to play a major role in cell-cell communication. For example, recent observations revealed the presence of diffusible factors with proliferative properties in the secretome of cancer cells. Thus, a qualitative and quantitative analysis of the secretome could lead to the identification of these factors and consequently to the development of new therapeutic strategies. Here, we provide an automated simple and effective strategy to identify novel targets in the secretome of specifically treated cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, we explore the supportive role of mass spectrometry (MS) in the development of functional assays of identified secreted target molecules. Simplicity is achieved by growing cells in medium free of serum, which eliminates the need to remove the most abundant serum proteins and at the same time reduces disturbing matrix effects. Upon identification of these factors, their validation and characterization will follow. Moreover, this approach can also lead to the identification of proteins abnormally secreted, shed, or oversecreted by cells as response to a stimulus. Furthermore, we also discuss the problems that one may encounter. Finally, we discuss the broad application of automated MS-based proteomics, particularly in cancer research, highlighting new horizons for the use of MS.

SCCRO (DCUN1D1) induces extracellular matrix invasion by activating matrix metalloproteinase 2.

PURPOSE: Ectopic expression of squamous cell carcinoma-related oncogene (SCCRO or DCUN1D1) in NIH-3T3 cells induces invasion in vitro and produces highly invasive xenografts in nude mice with a propensity for regional lymphatical metastasis. The aim of this study was to identify the molecular mechanism underlying SCCRO-induced invasion and metastasis. EXPERIMENTAL DESIGN: The molecular mechanism of SCCRO-mediated effects on matrix metalloproteinase-2 (MMP2) levels and activity were assessed using a combination of cell biological and molecular methods, including real-time PCR, reporter assay, RNA interference, and chromatin immunoprecipitation assay. Tumor specimens from primary upper aerodigestive tract carcinomas (n = 89) were examined for levels of SCCRO, MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2 mRNA by real-time PCR. RESULTS: Overexpression of SCCRO increases MMP2 levels and activity, which is required for SCCRO-induced invasion. Modified McKay assays reveal that SCCRO does not bind to the MMP2 promoter, suggesting that its transcriptional effects are indirect. Deletion or mutation of the activator protein-2 (AP2) and p53 binding element within the MMP2 promoter abrogates SCCRO-driven activation. Ectopic expression of SCCRO increases AP2 levels and promotes the binding of p53 to the MMP2 promoter. Consistent with these findings, SCCRO and MMP2 are coexpressed (P<0.0001; r(2)=0.58; 95% confidence interval, 0.46-0.69) in primary (upper aerodigestive tract) carcinomas (n=89), and this coexpression is associated with an increased prevalence of regional nodal metastasis (P=0.04; relative risk, 1.53). CONCLUSIONS: SCCRO-induced invasion involves activation of MMP2 transcription in an AP2- and p53-dependent manner. SCCRO is a potential marker for metastatic progression in affected cancers.

The role of novel oncogenes squamous cell carcinoma-related oncogene and phosphatidylinositol 3-kinase p110alpha in squamous cell carcinoma of the oral tongue.

PURPOSE: Amplification at chromosome 3q26.3 is a common and crucial event in head and neck squamous cell carcinoma (HNSCC), impacting significantly on tumor progression and clinical outcome. Two novel oncogenes, namely squamous cell carcinoma (SCC)-related oncogene (SCCRO) and PIK3CA (gene encoding phosphatidylinositol-3 kinase catalytic alpha-polypeptide), have been identified as targets of 3q26.3 amplification. This study aimed to delineate the role of SCCRO and PIK3CA in the pathogenesis of oral tongue SCC. EXPERIMENTAL DESIGN: The association between gene copy number for SCCRO and PIK3CA measured by fluorescence in situ hybridization and level of mRNA expression quantitated by real-time reverse transcription-PCR was assessed in a panel of human HNSCC cell lines. In addition, gene expression in 49 consecutive primary SCCs of the oral tongue was determined and correlated with clinicopathological characteristics and outcome. RESULTS: The mRNA level of SCCRO and PIK3CA was significantly correlated to the gene copy number in nine HNSCC cell lines. In addition, the expression level of SCCRO and PIK3CA was significantly greater in malignant tissues compared with those in histologically normal mucosae (2.17- and 2.46-fold, respectively; P < 0.001). Matched tumor normal control analysis revealed that 24.5 and 69.4% of patients expressed high levels of SCCRO and PIK3CA, respectively. Univariate analyses demonstrated that SCCRO overexpression correlated with nodal metastases (P = 0.05) and advanced stage (P = 0.02), whereas PIK3CA overexpression was associated with vascular invasion (P = 0.04). Only SCCRO overexpression was associated with disease-specific (P = 0.04) and overall survival (P = 0.02). Furthermore, SCCRO overexpression remained an independent predictor for cervical nodal metastasis on multivariate regression analysis (chi(2) likelihood ratio = 4.38; P = 0.04). CONCLUSIONS: Although both SCCRO and PIK3CA may play a role in the pathogenesis of oral tongue SCC through amplification at 3q26, SCCRO appears to be a significant predictor of regional metastasis and may be a marker for tumor aggressiveness and clinical outcome.

p53 regulates cell survival by inhibiting PIK3CA in squamous cell carcinomas.

Interactions between the p53 and PI3K/AKT pathways play a significant role in the determination of cell death/survival. In benign cells these pathways are interrelated through the transcriptional regulation of PTEN by p53, which is required for p53-mediated apoptosis. PTEN exerts its effects by decreasing the phosphorylated AKT fraction, thereby diminishing prosurvival activities. However, the link between these pathways in cancer is not known. In this study, PIK3CA, encoding the p110alpha catalytic subunit of PI3K, is identified as an oncogene involved in upper aerodigestive tract (UADT) carcinomas. Simultaneous abnormalities in both pathways are rare in primary tumors, suggesting that amplification of PIK3CA and mutation of p53 are mutually exclusive events and either event is able to promote a malignant phenotype. Moreover, the negative effect of p53 induction on cell survival involves the transcriptional inhibition of PIK3CA that is independent of PTEN activity, as PTEN is not expressed in the primary tumors. Conversely, constitutive activation of PIK3CA results in resistance to p53-related apoptosis in PTEN deficient cells. Thus, p53 regulates cell survival by inhibiting the PI3K/AKT prosurvival signal independent of PTEN in epithelial tumors. This inhibition is required for p53-mediated apoptosis in malignant cells.