An epigenetic auto-feedback loop regulates TGF-ß type II receptor expression and function in NSCLC.

The downregulation of transforming growth factor-ß (TGF-ß) type II receptor (TßRII) expression and function plays a pivotal role in the loss of the TGF-ß-induced tumor suppressor function that contributes to lung cancer progression. The aberrant expression of miRNAs has been shown to be involved in the regulation of oncogenes and tumor suppressor genes. Our current study involving miRNA microarray, northern blot and QRT-PCR analysis shows an inverse correlation between miR-20a and TßRII expression in non-small cell lung cancer (NSCLC) tissues and cell lines. Stable expression of miR-20a downregulates TßRII in lung epithelial cells which results in an inhibition of TGF-ß signaling and attenuation of TGF-ß-induced cell growth suppression and apoptosis. Stable knock down of miR-20a increases TßRII expression and inhibits tumorigenicity of lung cancer cells in vivo. Oncogene c-Myc promotes miR-20a expression by activating its promoter leading to downregulation of TßRII expression and TGF-ß signaling. MiR-145, which is upregulated by TGF-ß, inhibits miR-20a expression by targeting c-Myc and upregulates TßRII expression. These correlations among miRNAs and cellular proteins are supported by TCGA public database using NSCLC specimens. These results suggest a novel mechanism for the loss of TßRII expression and TGF-ß-induced tumor suppressor functions in lung cancer through a complex auto-feedback loop TGF-ß/miR-145/c-Myc/miR-20a/TßRII.

Elucidating the mechanism of regulation of transforming growth factor ß Type II receptor expression in human lung cancer cell lines.

Lung carcinogenesis in humans involves an accumulation of genetic and epigenetic changes that lead to alterations in normal lung epithelium, to in situ carcinoma, and finally to invasive and metastatic cancers. The loss of transforming growth factor ß (TGF-ß)-induced tumor suppressor function in tumors plays a pivotal role in this process, and our previous studies have shown that resistance to TGF-ß in lung cancers occurs mostly through the loss of TGF-ß type II receptor expression (TßRII). However, little is known about the mechanism of down-regulation of TßRII and how histone deacetylase (HDAC) inhibitors (HDIs) can restore TGF-ß-induced tumor suppressor function. Here we show that HDIs restore TßRII expression and that DNA hypermethylation has no effect on TßRII promoter activity in lung cancer cell lines. TGF-ß-induced tumor suppressor function is restored by HDIs in lung cancer cell lines that lack TßRII expression. Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by either activated Ras or epidermal growth factor signaling is involved in the down-regulation of TßRII through histone deacetylation. We have immunoprecipitated the protein complexes by biotinylated oligonucleotides corresponding to the HDI-responsive element in the TßRII promoter (-127/-75) and identified the proteins/factors using proteomics studies. The transcriptional repressor Meis1/2 is involved in repressing the TßRII promoter activity, possibly through its recruitment by Sp1 and NF-YA to the promoter. These results suggest a mechanism for the downregulation of TßRII in lung cancer and that TGF-ß tumor suppressor functions may be restored by HDIs in lung cancer patients with the loss of TßRII expression.

S-phase-coupled apoptosis in tumor suppression.

DNA replication is essential for accurate transmission of genomic information from parental to daughter cells. DNA replication is licensed once per cell division cycle. This process is highly regulated by both positive and negative regulators. Over-replication, under-replication, as well as DNA damage in a cell all induce the activation of checkpoint control pathways such as ATM/ATR, CHK kinases, and the tumor suppressor protein p53, which provide "damage controls" via either DNA repairs or apoptosis. This review focuses on accumulating evidence, with the emphasis on recently discovered Killin, that S-phase checkpoint control is crucial for a mammalian cell to make a life and death decision in order to safeguard genome integrity.

Linear dynamic range for signal detection in fluorescent differential display.

We have determined the linear dynamic range in signal detection by Fluorescent Differential Display (FDD) using conditionally induced mRNA expression of the p53 tumor-suppressor gene as a control. By serial spiking of p53-induced RNA into that of non-induced RNA, we were able to quantitatively measure up to 100-fold change in p53 mRNA expression level. The linear dynamic range of signal detection per mRNA message was determined to be from 1000 up to 20,000 in fluorescence signal, in which the signals for the majority of mRNAs reside. Thus, FDD can be used to accurately quantify differences in mRNA expression among eukaryotic cells.

Proof-reading signal accuracy of gene expression by binary differential display.

Differential display (DD) is commonly used for identifying differentially expressed genes. However, each cDNA species identified by DD must be verified so a "real difference" can be differentiated from false positives. Although Northern blot analysis is the gold standard it is labor intensive, time-consuming and requires a significant amount of RNA. To speed up and streamline the confirmation process, we developed a new strategy: binary differential display (BDD) based on the binding kinetics of the arbitrary primers in DD. After determining a cDNA sequence of interest from a DD screen, two more 13mer primers derived from the original arbitrary primer used can be designed to target a corresponding cDNA sequence of interest: one with perfect 5'-base matches and the other with additional mismatches at the 5'-base to the corresponding mRNA being confirmed. A separate reverse transcription and FDD are then performed with the same RNA samples being compared. BDD can quickly and accurately determine if a cDNA sequence identified by DD corresponds to a truly differentially expressed gene. In addition, the method is especially useful when more than one cDNA sequence was recovered from a DD band where the masking effect of false-positives can be clearly resolved. Given its simplicity and limited RNA sample required, BDD can be used as a general strategy for rapid confirmation of differentially expressed genes discovered by DD.

Automated fluorescent differential display for cancer gene profiling.

Since its invention in 1992, differential display (DD) has become the most commonly used technique for identifying differentially expressed genes because of its many advantages over competing technologies such as DNA microarray, serial analysis of gene expression (SAGE), and subtractive hybridization. A large number of these publications have been in the field of cancer, specifically on p53 target genes. Despite the great impact of the method on biomedical research, there had been a lack of automation of DD technology to increase its throughput and accuracy for systematic gene expression analysis. Many previous DD work has taken a "shotgun" approach of identifying one gene at a time, with a limited number of polymerase chain reactions (PCRs) set up manually, giving DD a low-tech and low-throughput image. We have optimized the DD process with a platform that incorporates fluorescent digital readout, automated liquid handling, and large-format gels capable of running entire 96-well plates. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. These major improvements will allow researchers to find differentially expressed genes of interest, both known and novel, quickly and easily.

Antimetastatic role of Smad4 signaling in colorectal cancer.

BACKGROUND & AIMS: Transforming growth factor (TGF)-beta signaling occurs through Smads 2/3/4, which translocate to the nucleus to regulate transcription; TGF-beta has tumor-suppressive effects in some tumor models and pro-metastatic effects in others. In patients with colorectal cancer (CRC), mutations or reduced levels of Smad4 have been correlated with reduced survival. However, the function of Smad signaling and the effects of TGF-beta-receptor kinase inhibitors have not been analyzed during CRC metastasis. We investigated the role of TGF-beta/Smad signaling in CRC progression. METHODS: We evaluated the role of TGF-beta/Smad signaling on cell proliferation, migration, invasion, tumorigenicity, and metastasis in Smad4-null colon carcinoma cell lines (MC38 and SW620) and in those that transgenically express Smad4. We also determined the effects of a TGF-beta-receptor kinase inhibitor (LY2109761) in CRC tumor progression and metastasis in mice. RESULTS: TGF-beta induced migration/invasion, tumorigenicity, and metastasis of Smad4-null MC38 and SW620 cells; incubation with LY2109761 reversed these effects. In mice, LY2109761 blocked metastasis of CRC cells to liver, inducing cancer cell expression of E-cadherin and reducing the expression of the tumorigenic proteins matrix metalloproteinase-9, nm23, urokinase plasminogen activator, and cyclooxygenase-2. Transgenic expression of Smad4 significantly reduced the oncogenic potential of MC38 and SW620 cells; in these transgenic cells, TGF-beta had tumor suppressor, rather than tumorigenic, effects. CONCLUSIONS: TGF-beta/Smad signaling suppresses progression and metastasis of CRC cells and tumors in mice. Loss of Smad4 might underlie the functional shift of TGF-beta from a tumor suppressor to a tumor promoter; inhibitors of TGF-beta signaling might be developed as CRC therapeutics.

Lipopolysaccharide-dependent interaction between PU.1 and c-Jun determines production of lipocalin-type prostaglandin D synthase and prostaglandin D2 in macrophages.

Previously, we reported that expression of lipocalin-prostaglandin D synthase (L-PGDS) is inducible in macrophages and protects from Pseudomonas pneumonia. Here, we investigated the mechanism by which L-PGDS gene expression is induced in macrophages. A promoter analysis of the murine L-PGDS promoter located a binding site of PU.1, a transcription factor essential for macrophage development and inflammatory gene expression. A chromatin immunoprecipitation assay showed that PU.1 bound to the cognate site in the endogenous L-PGDS promoter in response to LPS. Overexpression of PU.1, but not of PU.1(S148A), a mutant inert to casein kinase II (CKII) or NF-kappaB-inducing kinase (NIK), induced L-PGDS in RAW 264.7 cells. Conversely, siRNA silencing of PU.1 expression blunted productions of L-PGDS and prostaglandin D2 (PGD(2)). LPS treatment induced formation of the complex of PU.1 and cJun on the PU.1 site, but inactivation of cJun by treatment with JNK or p38 kinase inhibitor abolished the complex, and suppressed PU.1 transcriptional activity for L-PGDS gene expression. Together, these results show that PU.1, activated by CKII or NIK, cooperates with MAPK-activated cJun to maximally induce L-PGDS expression in macrophages following LPS treatment, and suggest that PU.1 participates in innate immunity through the production of L-PGDS and PGD(2).

Killin is a p53-regulated nuclear inhibitor of DNA synthesis.

Cell growth arrest and apoptosis are two best-known biological functions of tumor-suppressor p53. However, genetic evidence indicates that not only is p21 the major mediator of G(1) arrest, but also it can prevent apoptosis with an unknown mechanism. Here, we report the discovery of a p53 target gene dubbed killin, which lies in close proximity to pten on human chromosome 10 and encodes a 20-kDa nuclear protein. We show that Killin is not only necessary but also sufficient for p53-induced apoptosis. Genetic and biochemical analysis demonstrates that Killin is a high-affinity DNA-binding protein, which potently inhibits eukaryotic DNA synthesis in vitro and appears to trigger S phase arrest before apoptosis in vivo. The DNA-binding domain essential for DNA synthesis inhibition was mapped to within 42 amino acid residues near the N terminus of Killin. These results support Killin as a missing link between p53 activation and S phase checkpoint control designed to eliminate replicating precancerous cells, should they escape G(1) blockade mediated by p21.

CYFIP2, a direct p53 target, is leptomycin-B sensitive.

A number of target genes for the tumor suppressor, p53, have been identified, however, the mechanisms that contribute to p53-dependent apoptosis remain to be fully elucidated. In a comprehensive screen for p53 target genes, we have identified Cytoplasmic FMR Interacting Protein 2 (CYFIP2) as a p53-inducible gene. Here we show that the CYFIP2 promoter contains a p53-responsive element that confers p53 binding as well as transcriptional activation of a heterologous reporter. Inducible expression of CYFIP2 is sufficient for caspase activation and cellular apoptosis, reminiscent of p53 activation. Together, these results suggest that CYFIP2 is a direct p53 target gene that may be part of a redundant network of genes responsible for p53-dependent apoptosis. In addition, the sensitivity of CYFIP2 protein subcellular localization to Leptomycin-B, a CRM-1/Exportin inhibitor, suggests that the biological functions of CYFIP2 may extend from the cytoplasmic compartment into the nucleus of the cell.

TIS11D is a candidate pro-apoptotic p53 target gene.

A number of target genes for the tumor suppressor, p53, have been identified, however, the mechanisms that contribute to p53-dependent apoptosis remain to be fully elucidated. In a comprehensive screen for p53 target genes by differential display, we have identified TIS11D as a p53-inducible gene. Induction of TIS11D mRNA was confirmed by Northern Blot in response to p53 expression. Inducible expression of TIS11D resulted in inhibition of cell proliferation and apoptosis. These data suggest TIS11D as a candidate p53 target gene that may be part of the network of genes responsible for p53-dependent apoptosis.

Automation of fluorescent differential display with digital readout.

Since its invention in 1992, differential display (DD) has become the most commonly used technique for identifying differentially expressed genes because of its many advantages over competing technologies such as DNA microarray, serial analysis of gene expression (SAGE), and subtractive hybridization. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for systematic gene expression analysis. Most of previous DD work has taken a "shot-gun" approach of identifying one gene at a time, with a limited number of polymerase chain reaction (PCR) reactions set up manually, giving DD a low-tech and low-throughput image. We have optimized the DD process with a new platform that incorporates fluorescent digital readout, automated liquid handling, and large-format gels capable of running entire 96-well plates. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. These major improvements will allow researchers to find differentially expressed genes of interest, both known and novel, quickly and easily.

Saturation screening for p53 target genes by digital fluorescent differential display.

Differential display (DD) is one of the most commonly used approaches for identifying differentially expressed genes. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for a systematic gene expression analysis. Most of previous DD work has taken a "shotgun" approach of identifying one gene at a time, with limited polymerase chain reaction (PCR) reactions set up manually, giving DD a low-technology and low-throughput image. With our newly created DD mathematical model, which has been validated by computer simulations, global analysis of gene expression by DD technology is no longer a shot in the dark. After identifying the "rate-limiting" factors that contribute to the "noise" level of DD method, we have optimized the DD process with a new platform that incorporates fluorescent digital readout and automated liquid handling. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. We are using this newly integrated FDD technology to conduct a systematic and comprehensive screening for p53 tumor-suppressor gene targets.

Identification of p53 target genes by fluorescent differential display.

Differential display (DD) is a method used worldwide for identifying differentially expressed genes in eukaryotic cells. The mRNA DD technology works by systematic amplification of the 3' terminal regions of mRNAs. Using anchored primers designed to bind 5' boundary of the polyA tails for reverse transcription, followed by polymerase chain reaction (PCR) amplification with additional upstream primers of arbitrary sequences, mRNA subpopulations are separated by denaturing polyacrylamide electrophoresis. This allows direct side-by-side comparison of most of the mRNAs between or among related cells. Because of its simplicity, sensitivity, and reproducibility, the mRNA DD method is finding wide-ranging and rapid applications in developmental biology, cancer research, neuroscience, pathology, endocrinology, plant physiology, and many other areas. Since the recent development of the fluorescent differential display (FDD), the first nonradioactive DD system with equivalent sensitivity to the original 33P isotopic labeling method, it is now possible with this technology to automate, which can greatly increase the throughput and accuracy of mRNA DD.

Systematic analysis of intrinsic factors affecting differential display.

Differential display (DD) is a widely used method for identifying differentially expressed genes. To improve further the efficiency and reproducibility of the method, this report systematically examines four critical parameters of standard DD-PCR. Specifically, the study determined the optimal annealing temperature, elongation time, dNTP concentration, and arbitrary primer concentration. By using a thermal cycler that was capable of displaying a temperature gradient across a PCR plate, it was possible to determine (in a single experiment) the effect of different annealing temperatures. The optimal annealing temperaturefor a 13-mer arbitrary primer fell within a broad range of 40 degrees C-50 degrees C. Elongation times over a range of 30-120 s worked best. The optimal concentration for dNTPs was within a very broad range of 2-50 microM, with higher amounts allowing for greater pipetting accuracy. The most favorable concentration for the arbitrary primer was also within a broad range of 0.1-2.0 microM. A primer concentration below this range greatly reduced the efficiency of the amplification process. In conclusion, the experimental findings delineated the best possible DD conditions for a more reliable assessment of differential gene expression.