SummaryNatural genetic transformation in Streptococcus pneumoniae is controlled in part by a quorum-sensing system mediated by a peptide pheromone called competence-stimulating peptide (CSP), which acts to coordinate transient activation of genes required for competence. To characterize the transcriptional response and regulatory events occurring when cells are exposed to competence pheromone, we constructed DNA microarrays and analysed the temporal expression profiles of 1817 among the 2129 unique predicted open reading frames present in the S. pneumoniae TIGR4 genome (84%). After CSP stimulation, responsive genes exhibited four temporally distinct expression profiles: early, late and delayed gene induction, and gene repression. At least eight early genes participate in competence regulation including comX , which encodes an alternative sigma factor. Late genes were dependent on ComX for CSPinduced expression, many playing important roles in transformation. Genes in the delayed class (third temporal wave) appear to be stress related. Genes repressed during the CSP response include ribosomal protein loci and other genes involved in protein synthesis. This study increased the number of identified CSP-responsive genes from approximately 40 to 188. Given the relatively large number of induced genes (6% of the genome), it was of interest to determine which genes provide functions essential to transformation. Many of the induced loci were subjected to gene disruption mutagenesis, allowing us to establish that among 124 CSP-inducible genes, 67 were individually dispensable for transformation, whereas 23 were required for transformation.

Recent studies have shown that there is a considerable heterogeneity in the response of melanoma cell lines to MEK and BRAF inhibitors. In the current study, we address whether dysregulation of cyclin-dependent kinase 4 (CDK4) and/or cyclin D1 contribute to the BRAF inhibitor resistance of melanoma cells. Mutational screening identified a panel of melanoma cell lines that harbored both a BRAF V600E mutation and a CDK4 mutation: K22Q (1205Lu), R24C (WM39, WM46, and SK-Mel-28), and R24L (WM902B). Pharmacologic studies showed that the presence of a CDK4 mutation did not alter the sensitivity of these cell lines to the BRAF inhibitor. The only cell line with significant BRAF inhibitor resistance was found to harbor both a CDK4 mutation and a CCND1 amplification. Array comparative genomic hybridization analysis showed that CCND1 was amplified in 17% of BRAF V600E -mutated human metastatic melanoma samples, indicating the clinical relevance of this finding. As the levels of CCND1 amplification in cell lines are lower than those seen in clinical specimens, we overexpressed cyclin D1 alone and in the presence of CDK4 in a drug-sensitive melanoma line. Cyclin D1 overexpression alone increased resistance and this was enhanced when cyclin D1 and CDK4 were concurrently overexpressed. In conclusion, increased levels of cyclin D1, resulting from genomic amplification, may contribute to the BRAF inhibitor resistance of BRAF V600E -mutated melanomas, particularly when found in the context of a CDK4 mutation/ overexpression. [Mol Cancer Ther 2008;7(9):2876 -83]

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Regulation of classic cadherins plays a critical role in tissue remodeling during development and cancer; however, less attention has been paid to the importance of desmosomal cadherins. We previously showed that EGFR inhibition results in accumulation of the desmosomal cadherin, desmoglein 2 (Dsg2), at cell-cell interfaces accompanied by inhibition of matrix metalloprotease (MMP)-dependent shedding of the Dsg2 ectodomain and tyrosine phosphorylation of its cytoplasmic domain. Here, we show that EGFR inhibition stabilizes Dsg2 at intercellular junctions by interfering with its accumulation in an internalized cytoplasmic pool. Furthermore, MMP inhibition and ADAM17 RNAi, blocked shedding and depleted internalized Dsg2, but less so E-cadherin, in highly invasive SCC68 cells. ADAM9 and 15 silencing also impaired Dsg2 processing, supporting the idea that this desmosomal cadherin can be regulated by multiple ADAM family members. In contrast, ADAM10 siRNA enhanced accumulation of a 100-kDa Dsg2 cleavage product and internalized pool of Dsg2. Although both MMP and EGFR inhibition increased intercellular adhesive strength in control cells, the response to MMP-inhibition was Dsg2-dependent. These data support a role for endocytic trafficking in regulating desmosomal cadherin turnover and function and raise the possibility that internalization and regulation of desmosomal and classic cadherin function can be uncoupled mechanistically. INTRODUCTIONThe ability of cells to modulate their contacts with each other and the underlying matrix is essential for epithelial remodeling that occurs in development and cancer progression (Behrens, 1999;Thiery, 2003;Kramer et al., 2005). In particular, members of cadherin family of calcium-dependent intercellular adhesion molecules have been demonstrated to both suppress (Frixen et al., 1991) and promote (Islam et al., 1996) cell migration and invasion. Although classic cadherins assemble into intercellular adhesive structures known as adherens junctions that associate with the cortical actin cytoskeleton, desmosomal cadherins, including desmogleins and desmocollins, make up the adhesive core of desmosomes, which anchor intermediate filaments (IF) to sites of strong intercellular adhesion (Green and Simpson, 2007).Anchorage to the IF cytoskeleton is established with the cooperation of the desmosomal cadherin-associated proteins plakoglobin and plakophilins, which in turn link the IF-associated protein desmoplakin (DP) to the membrane complex. Desmosomes provide mechanical integrity to epithelial and heart tissues by redistributing the forces of mechanical stress . In addition, desmosomal cadherins have more recently emerged as playing a role in tissue morphogenesis (Runswick et al., 2001;Chidgey and Dawson, 2007;Dusek et al., 2007).In spite of desmosomes' importance in tissue function, most studies have focused on the contribution of classic cadherins to epithelial remodeling. Classic cadherins engage in bidirectional signaling with receptor tyrosine kinases (RTKs), whereby they are both ...

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SummaryWe previously showed that the cell-cell junction protein plakoglobin (PG) not only suppresses motility of keratinocytes in contact with each other, but also, unexpectedly, of single cells. Here keratinocytes. PG -/-cells also exhibited Src-independent activation of the small GTPases Rac1 and RhoA. Both Src and RhoA inhibition attenuated PG -/-keratinocyte motility. We propose a novel role for PG in regulating cell motility through distinct ECM-Src and RhoGTPase-dependent pathways, influenced in part by PG-dependent regulation of FN mRNA stability. Key words: Armadillo protein, Desmosome, Extracellular matrix, KeratinocyteJournal of Cell Science (Caldelari et al., 2001) and adhesive strength (Acehan et al., 2008) in keratinocytes lacking PG. Moreover, PG-null mice and patients with pathogenic homozygous PG mutations have impaired tissue integrity associated with skin and heart defects (Aberle et al., 1995;Bierkamp et al., 1996;McKoy et al., 2000;Ruiz et al., 1996). PG is also found in the cytoplasm and nucleus (Green and Simpson, 2007;Schmidt and Koch, 2007), where it is able to act independently of its function in intercellular adhesion. Its adhesionindependent functions are still not well defined, but the data suggest that PG can regulate gene expression and protein stability (Aktary et al., 2010;Hakimelahi et al., 2000;Shimizu et al., 2008) in both a -catenin-dependent and -independent manner (Raurell et al., 2006;Teuliere et al., 2004;Yin and Green, 2004;Zhurinsky et al., 2000).Recently we demonstrated that PG not only inhibits motility of keratinocytes in contact, but also inhibits Src-dependent single cell motility (Yin et al., 2005b). The observed changes in motility and altered cell morphology of PG -/-keratinocytes suggested to us that PG could be regulating cell-substrate interactions by modulating components of the extracellular matrix (ECM), its integrin receptors and/or the molecules involved in ECM-triggered motility cues. Using a combination of live cell imaging and cross plating, we show here that PG expression has a potent impact on the organization of actin, its associated membrane protrusions, focal adhesions and Src-dependent motility, in large part through regulation of the expression levels of the underlying ECM components. In particular, the ability of PG to regulate fibronectin (FN; also know as Fn1) mRNA stability was identified as a novel mechanism contributing to PG-dependent suppression of keratinocyte motility. Further analysis indicated that PG-dependent alterations in activity of the small GTPases Rac1 and RhoA act in parallel with FN/Src-dependent regulation of cortical actin 3577 Plakoglobin regulates motility via ECM structures to fine tune the motile behavior of keratinocytes. Collectively, these results indicate that a desmosomal molecule, PG, is capable of regulating single cell motility through matrix deposition in concert with Rho GTPases, independently of its role as a cell-cell adhesion molecule. Results PG regulates keratinocyte cell polarity and single-cell ...

This study aimed to evaluate whether patients with advanced non-small-cell lung cancer experience disrupted rest -activity daily rhythms, poor sleep quality, weakness, and maintain attributes that are linked to circadian function such as fatigue. This report describes the rest -activity patterns of 33 non-small-cell lung cancer patients who participated in a randomised clinical trial evaluating the benefits of melatonin. Data are reported on circadian function, health-related quality of life (QoL), subjective sleep quality, and anxiety/depression levels prior to randomisation and treatment. Actigraphy data, an objective measure of circadian function, demonstrated that patients' rest -activity circadian function differs significantly from control subjects. Our patients reported poor sleep quality and high levels of fatigue. Ferrans and Powers QoL Index instrument found a high level of dissatisfaction with healthrelated QoL. Data from the European Organization for Research and Treatment for Cancer reported poor capacity to fulfil the activities of daily living. Patients studied in the hospital during or near chemotherapy had significantly more abnormal circadian function than those studied in the ambulatory setting. Our data indicate that measurement of circadian sleep/activity dynamics should be accomplished in the outpatient/home setting for a minimum of 4 -7 circadian cycles to assure that they are most representative of the patients' true condition. We conclude that the daily sleep/activity patterns of patients with advanced lung cancer are disturbed. These are accompanied by marked disruption of QoL and function. These data argue for investigating how much of this poor functioning and QoL are actually caused by this circadian disruption, and, whether behavioural, light-based, and or pharmacologic strategies to correct the circadian/sleep activity patterns can improve function and QoL.

Prostate cancer mortality is primarily attributed to metastatic rather than primary, organ-confined disease. Acquiring a motile and invasive phenotype is an important step in development of tumors and ultimately metastasis. This step involves remodeling of the extracellular matrix and of cell-matrix interactions, cell movement mediated by the actin cytoskeleton, and activation of focal adhesion kinase (FAK)/Src signaling. Epidemiologic studies suggest that the metastatic behavior of prostate cancer may be an ideal target for chemoprevention. The natural flavone apigenin is known to have chemopreventive properties against many cancers, including prostate cancer. Here, we study the effect of apigenin on motility, invasion, and its mechanism of action in metastatic prostate carcinoma cells (PC3-M). We found that apigenin inhibits PC3-M cell motility in a scratchwound assay. Live cell imaging studies show that apigenin diminishes the speed and affects directionality of cell motion. Alterations in the cytoskeleton are consistent with impaired cell movement in apigenin-treated cells. Apigenin treatment leads to formation of "exaggerated filopodia," which show accumulation of focal adhesion proteins at their tips. Furthermore, apigenin-treated cells adhere more strongly to the extracellular matrix. Additionally, apigenin decreases activation of FAK and Src, and phosphorylation of Src substrates FAK Y576/577 and Y925. Expression of constitutively active Src blunts the effect of apigenin on cell motility and cytoskeleton remodeling. These results show that apigenin inhibits motility and invasion of prostate carcinoma cells, disrupts actin cytoskeleton organization, and inhibits FAK/Src signaling. These studies provide mechanistic insight into developing novel strategies for inhibiting prostate cancer cell motility and invasiveness.Prostate cancer is the most common noncutaneous malignancy in American males (over 186,000 cases diagnosed yearly), and the second leading cause of cancer-related deaths in American men (28,660 estimated deaths in 2008; ref. 1). In prostate cancer, most deaths are attributed to metastatic disease rather than primary, organ-confined prostate cancer (2-5). Numerous epidemiologic studies (6) suggest that the metastatic behavior of prostate cancer may be an ideal target for pharmacologic intervention with chemopreventive agents. Because prostate cancer is typically diagnosed in older men, chemopreventive and/or chemotherapeutic strategies to delay its progression may have a substantial impact on clinical outcome. Epidemiologic studies have shown that Asian men experience a lower incidence of metastatic prostate cancer than Western men, which is attributed to dietary and/or life-style factors (3, 4). Interestingly, some studies suggest that the incidence of primary cancer may be similar in Asian and Western populations, and that the higher rate of prostate cancer-related deaths in the United States is due to a higher rate of prostate cancer metastasis (3,5).Apigenin (4′, 5, 7-trihydroxyflavo...