Paraoxonase 2 Facilitates Pancreatic Cancer Growth and Metastasis by Stimulating GLUT1-Mediated Glucose Transport.

Metabolic deregulation is a hallmark of human cancers, and the glycolytic and glutamine metabolism pathways were shown to be deregulated in pancreatic ductal adenocarcinoma (PDAC). To identify new metabolic regulators of PDAC tumor growth and metastasis, we systematically knocked down metabolic genes that were overexpressed in human PDAC tumor samples using short hairpin RNAs. We found that p53 transcriptionally represses paraoxonase 2 (PON2), which regulates GLUT1-mediated glucose transport via stomatin. The loss of PON2 initiates the cellular starvation response and activates AMP-activated protein kinase (AMPK). In turn, AMPK activates FOXO3A and its transcriptional target, PUMA, which induces anoikis to suppress PDAC tumor growth and metastasis. Pharmacological or genetic activation of AMPK, similar to PON2 inhibition, blocks PDAC tumor growth. Collectively, our results identify PON2 as a new modulator of glucose transport that regulates a pharmacologically tractable pathway necessary for PDAC tumor growth and metastasis.

Loss of thymidine kinase 1 inhibits lung cancer growth and metastatic attributes by reducing GDF15 expression.

Metabolic alterations that are critical for cancer cell growth and metastasis are one of the key hallmarks of cancer. Here, we show that thymidine kinase 1 (TK1) is significantly overexpressed in tumor samples from lung adenocarcinoma (LUAD) patients relative to normal controls, and this TK1 overexpression is associated with significantly reduced overall survival and cancer recurrence. Genetic knockdown of TK1 with short hairpin RNAs (shRNAs) inhibits both the growth and metastatic attributes of LUAD cells in culture and in mice. We further show that transcriptional overexpression of TK1 in LUAD cells is driven, in part, by MAP kinase pathway in a transcription factor MAZ dependent manner. Using targeted and gene expression profiling-based approaches, we then show that loss of TK1 in LUAD cells results in reduced Rho GTPase activity and reduced expression of growth and differentiation factor 15 (GDF15). Furthermore, ectopic expression of GDF15 can partially rescue TK1 knockdown-induced LUAD growth and metastasis inhibition, confirming its important role as a downstream mediator of TK1 function in LUAD. Collectively, our findings demonstrate that TK1 facilitates LUAD tumor and metastatic growth and represents a target for LUAD therapy.

Senescence induction in human fibroblasts and hematopoietic progenitors by leukemogenic fusion proteins.

Hematologic malignancies are typically associated with leukemogenic fusion proteins, which are required to maintain the oncogenic state. Previous studies have shown that certain oncogenes that promote solid tumors, such as RAS and BRAF, can induce senescence in primary cells, which is thought to provide a barrier to tumorigenesis. In these cases, the activated oncogene elicits a DNA damage response (DDR), which is essential for the senescence program. Here we show that 3 leukemogenic fusion proteins, BCR-ABL, CBFB-MYH11, and RUNX1-ETO, can induce senescence in primary fibroblasts and hematopoietic progenitors. Unexpectedly, we find that senescence induction by BCR-ABL and CBFB-MYH11 occurs through a DDR-independent pathway, whereas RUNX1-ETO induces senescence in a DDR-dependent manner. All 3 fusion proteins activate the p38 MAPK pathway, which is required for senescence induction. Our results reveal diverse pathways for oncogene-induced senescence and further suggest that leukemias harbor genetic or epigenetic alterations that inactivate senescence induction genes.

TolA mediates the differential detergent resistance pattern between the Salmonella enterica subsp. enterica serovars Typhi and Typhimurium.

The tol-pal genes are essential for maintaining the outer membrane integrity and detergent resistance in various Gram-negative bacteria, including Salmonella. The role of TolA has been well established for the bile resistance of Salmonella enterica subsp. enterica serovar Typhimurium. We compared the bile resistance pattern between the S. enterica serovars Typhi and Typhimurium and observed that Typhi is more resistant to bile-mediated damage. A closer look revealed a significant difference in the TolA sequence between the two serovars which contributes to the differential detergent resistance. The tolA knockout of both the serovars behaves completely differently in terms of membrane organization and morphology. The role of the Pal proteins and difference in LPS organization between the two serovars were verified and were found to have no direct connection with the altered bile resistance. In normal Luria broth (LB), S. Typhi ΔtolA is filamentous while S. Typhimurium ΔtolA grows as single cells, similar to the wild-type. In low osmolarity LB, however, S. Typhimurium ΔtolA started chaining and S. Typhi ΔtolA showed no growth. Further investigation revealed that the chaining phenomenon observed was the result of failure of the outer membrane to separate in the dividing cells. Taken together, the results substantiate the evolution of a shorter TolA in S. Typhi to counteract high bile concentrations, at the cost of lower osmotic tolerance.

N-acylsphingosine amidohydrolase 1 promotes melanoma growth and metastasis by suppressing peroxisome biogenesis-induced ROS production.

OBJECTIVE: Metabolic deregulation is a key hallmark of cancer cells and has been shown to drive cancer growth and metastasis. However, not all metabolic drivers of melanoma are known. Based on our finding that N-acylsphingosine amidohydrolase 1 (ASAH1) is overexpressed in melanoma, the objective of these studies was to establish its role in melanoma tumor growth and metastasis, understand its mechanism of action, and evaluate ASAH1 targeting for melanoma therapy. METHODS: We used publicly available melanoma datasets and patient-derived samples of melanoma and normal skin tissue and analyzed them for ASAH1 mRNA expression and ASAH1 protein expression using immunohistochemistry. ASAH1 was knocked down using short-hairpin RNAs in multiple melanoma cell lines that were tested in a series of cell culture-based assays and mouse-based melanoma xenograft assays to monitor the effect of ASAH1 knockdown on melanoma tumor growth and metastasis. An unbiased metabolomics analysis was performed to identify the mechanism of ASAH1 action. Based on the metabolomics findings, the role of peroxisome-mediated reactive oxygen species (ROS) production was explored in regard to mediating the effect of ASAH1. The ASAH1 inhibitor was used alone or in combination with a BRAFV600E inhibitor to evaluate the therapeutic value of ASAH1 targeting for melanoma therapy. RESULTS: We determined that ASAH1 was overexpressed in a large percentage of melanoma cells and regulated by transcription factor E2F1 in a mitogen-activated protein (MAP) kinase pathway-dependent manner. ASAH1 expression was necessary to maintain melanoma tumor growth and metastatic attributes in cell cultures and mouse models of melanoma tumor growth and metastasis. To identify the mechanism by which ASAH1 facilitates melanoma tumor growth and metastasis, we performed a large-scale and unbiased metabolomics analysis of melanoma cells expressing ASAH1 short-hairpin RNAs (shRNAs). We found that ASAH1 inhibition increased peroxisome biogenesis through ceramide-mediated PPARγ activation. ASAH1 loss increased ceramide and peroxisome-derived ROS, which in turn inhibited melanoma growth. Pharmacological inhibition of ASAH1 also attenuated melanoma growth and enhanced the effectiveness of BRAF kinase inhibitor in the cell cultures and mice. CONCLUSIONS: Collectively, these results demonstrate that ASAH1 is a druggable driver of melanoma tumor growth and metastasis that functions by suppressing peroxisome biogenesis, thereby inhibiting peroxisome-derived ROS production. These studies also highlight the therapeutic utility of ASAH1 inhibitors for melanoma therapy.

Reliable means of diagnosis and serovar determination of blood-borne Salmonella strains: quick PCR amplification of unique genomic loci by novel primer sets.

Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR-based diagnosis method by designing primers against a region that is unique to Salmonella enterica subsp. enterica serovar Typhi and Salmonella enterica subsp. enterica serovar Paratyphi A, corresponding to the STY0312 gene in S. Typhi and its homolog SPA2476 in S. Paratyphi A. An additional set of primers amplify another region in S. Typhi CT18 and S. Typhi Ty2 corresponding to the region between genes STY0313 to STY0316 but which is absent in S. Paratyphi A. The possibility of a false-negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella serovars as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying the region from clinical isolates of patients from various geographical locations in India, thereby showing that this region is potentially stable. These set of primers can also differentiate between S. Typhi CT18, S. Typhi Ty2, and S. Paratyphi A, which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivity of 95% compared to the Widal test which has a sensitivity of only 63%. As observed, in certain cases, the PCR assay was more sensitive than the blood culture test was, as the PCR-based detection could also detect dead bacteria.

DNA Transfection Mediated by Cationic Lipid Reagents.

Liposomal transfection reagents vary in their ability to transfect cell lines efficiently. Some are generalists, whereas others are best used with specific cell types. The nonliposomal FuGENE 6 and the cationic liposomal Lipofectamine 2000 are examples of reagents that can successfully transfect most adherent and suspension cell types (including several primary and hard-to-transfect cell types) with negligible toxicity and a minimal number of manipulations. Importantly, both reagents can be used to transfect cells in the presence of serum, minimizing the number of manipulations during the transfection procedure. We also provide an alternative protocol that uses the cationic lipid reagents Lipofectin and Transfectam.

Histochemical Staining of Cell Monolayers for ß-Galactosidase.

There are several methods for assaying the success of transient transfections. If a plasmid expressing Escherichia coli ß-galactosidase was used, then this histochemical staining procedure is simple to perform and yields dependable results. The following method was designed for cells growing in 60-mm culture dishes.

Lipofection.

In the past, cloned DNA was introduced into cultured eukaryotic cells chiefly by biochemical methods using either calcium phosphate or diethylaminoethyl (DEAE)-dextran. Lipid reagents are now preferred because of the high efficiency of transfection that can be obtained and because of the ability of this class of reagents to mediate transfection of all types of nucleic acids into a wide range of cell types.

Introducing Genes into Cultured Mammalian Cells.

Strategies for the delivery of genes into eukaryotic cells fall into three categories: transfection by biochemical methods, transfection by physical methods, and virus-mediated transduction. This introduction deals with the first two categories.

DNA Transfection by Electroporation.

Electroporation, which uses pulsed electrical fields, can be used to introduce DNA into a variety of animal cells, plant cells, and bacteria. Electroporation works well with cell lines that are refractory to other transfection techniques, such as lipofection and calcium phosphate-DNA coprecipitation. But, as with other transfection methods, the optimal conditions for electroporating DNA into untested cell lines must be determined empirically. Several different electroporation instruments are available commercially, and manufacturers supply detailed protocols for their use with specific cell types and guidelines for optimization with others. This method describes the conditions for electroporating mammalian cell lines using the Gene Pulser Xcell Electroporation System (Bio-Rad).

Electroporation.

Electroporation is a process in which brief electrical pulses create transient pores in the plasma membrane that allow nucleic acids to enter the cellular cytoplasm. Here, we provide information on the history, mechanism, and optimization of electroporation. We also describe nucleofection, an improvement of the electroporation technology that permits the introduction of nucleic acids directly into the nucleus.

Calcium Phosphate-Mediated Transfection of Eukaryotic Cells with Plasmid DNAs.

This protocol describes a calcium phosphate-mediated transfection method for use with plasmid DNAs and adherent cells. At the end of the protocol is an alternative method for high-efficiency generation of stable transfectants.

Calcium Phosphate-Mediated Transfection of Cells with High-Molecular-Weight Genomic DNA.

This protocol describes a calcium phosphate-mediated transfection method for use with high-molecular-weight genomic DNAs.

Calcium Phosphate-Mediated Transfection of Adherent Cells or Cells Growing in Suspension: Variations on the Basic Method.

This protocol describes two variations of the calcium phosphate-mediated transfection method. The first can be used with all types of adherent cells, but is particularly useful for polarized epithelial cells, which do not efficiently take up material by endocytosis through the apical plasma membrane. To improve transfection efficiency, adherent cells are trypsinized and collected by centrifugation. The cells are resuspended in the calcium phosphate-DNA coprecipitate and then plated again on tissue culture dishes. Most lines of cells grown in suspension are resistant to calcium phosphate-mediated transfection methods; however, a few cell lines grown as suspension cultures (e.g., HeLa cells) can be transfected using the second modified calcium phosphate procedure described here.

Transfection of Mammalian Cells with Calcium Phosphate-DNA Coprecipitates.

Biochemical methods of transfection, including calcium phosphate-mediated and diethylaminoethyl (DEAE)-dextran-mediated transfection, have been used for many years to deliver nucleic acids into cultured cells. Here, we briefly review the use of calcium phosphate-DNA coprecipitates for transfection.

Dot Blot Analysis for Measuring Global N6-Methyladenosine Modification of RNA.

Posttranscriptional modification of mRNAs plays an important role in establishing the functional diversity of the proteome. The m6A modification is found in many species of RNA, including tRNA, mRNA, rRNA, and long noncoding RNAs. The physiological role of m6A modification of RNA is not fully explored and is a topic of current research. It is predicted that the major effect of m6A modification of mRNAs is on its stability and/or translation. The global changes in m6A levels in total RNA or particular species of RNAs can be measured by dot blot analysis using m6A specific antibodies or using mass spectrometry following chromatographic separation. The dot blot method for detection of global m6A changes is a relatively straightforward method to quantitate m6A modification but suffers from low sensitivity when the fraction of m6A-modified RNA is small in analyzed samples. Here, we describe a modified dot blot method that is sensitive and quantitative for detecting m6A-modified RNA by adding an immunoprecipitation step to enrich for m6A-modified RNA.

Selective Agents for Stable Transfection.

A stable cell line is generated when transfected DNA undergoes integration into a chromosome by nonhomologous recombination. Cells that stably express selectable (e.g., antibiotic-resistant) markers are also likely to have incorporated other DNA sequences. This phenomenon, in which physically unlinked genes are assembled into a single integrated array and expressed in the same transfected cell, is known as "cotransfection." Resistance to antibiotics has proven to be effective in selecting cotransfectants and, in some cases, as a driver for gene amplification.

Optical Transfection.

Strategies for the delivery of genes into eukaryotic cells fall into three categories: transfection by biochemical methods, transfection by physical methods, and virus-mediated transduction. "Optical transfection"-a physical transfection method-exploits the ability of light to create small transient pores in the plasma membrane of mammalian cells.

Analysis of Cell Viability by the alamarBlue Assay.

This protocol describes viability measurements for cell cultures in a 96-well tissue culture plate using alamarBlue (resazurin). The assay can be modified to accommodate larger plates; however, for a preliminary analysis of transfection reagents and parameters of the transfection protocol on cell viability, a 96-well plate format is the most cost effective.