Background Non-small cell lung cancers (NSLC) are aggressive cancers that are insensitive to chemotherapies and accounts for nearly 33% of all cancer deaths in the United States. Two hallmarks of cancer that allow cells to invade and metastasize are sustained proliferation and enhanced motility. In this study we investigate the relationship between urokinase plasminogen activator (uPA)/uPA receptor (uPAR) signaling and Na+/H+ exchanger isoform 1 (NHE1) expression and activity. Methods and Results The addition of 10nM uPA increased the carcinogenic potential of three NSCLC cell lines, NCI-H358, NCI-H460, and NCI-H1299. This included an increase in the rate of cell proliferation 1.6 to 1.9 fold; an increase in the percentage of cells displaying stress fibers 3.05 to 3.17 fold; and an increase in anchorage-independent growth from 1.64 to 2.0 fold. In each of these cases the increase was blocked when the experiments were performed with NHE1 inhibited by 10 μM EIPA (ethylisopropyl amiloride). To further evaluate the role of uPA/uPAR and NHE1 in tumor progression we assessed signaling events using full-length uPA compared to the uPA amino terminal fragment (ATF). Comparing uPA and ATF signaling in H460 cells, we found that both uPA and ATF increased stress fiber formation approximately 2 fold, while uPA increased matrix metalloproteinase 9 (MMP9) activity 5.44 fold compared to 2.81 fold for ATF. To expand this signaling study, two new cell lines were generated, one with reduced NHE1 expression (H460 NHE1 K/D) and one with reduced uPAR expression (H460 uPAR K/D). Using the K/D cell lines we found that neither uPA nor ATF could stimulate stress fiber formation or MMP9 activity in cells with dramatically decreased NHE1 or uPAR expression. Finally, using in vivo tumor formation studies in athymic mice we found that when mice were injected with H460 cells 80% of mice formed tumors with an average volume of 390 mm3. This was compared to 20% of H460 uPAR K/D injected mice forming tumors with an average volume of 15 mm3 and 10% of H460 NHE1 K/D injected mice forming tumors with an average volume of 5 mm3. Conclusion Taken together, these data demonstrate that uPA/uPAR-mediated tumor progression and metastasis requires NHE1 in NSCLC cells and suggests a potential therapeutic approach to blocking cancer progression.
BackgroundCultures of human proximal tubule cells have been widely utilized to study the role of EMT in renal disease. The goal of this study was to define the role of growth media composition on classic EMT responses, define the expression of E- and N-cadherin, and define the functional epitope of MT-3 that mediates MET in HK-2 cells.MethodsImmunohistochemistry, microdissection, real-time PCR, western blotting, and ELISA were used to define the expression of E- and N-cadherin mRNA and protein in HK-2 and HPT cell cultures. Site-directed mutagenesis, stable transfection, measurement of transepithelial resistance and dome formation were used to define the unique amino acid sequence of MT-3 associated with MET in HK-2 cells.ResultsIt was shown that both E- and N-cadherin mRNA and protein are expressed in the human renal proximal tubule. It was shown, based on the pattern of cadherin expression, connexin expression, vectorial active transport, and transepithelial resistance, that the HK-2 cell line has already undergone many of the early features associated with EMT. It was shown that the unique, six amino acid, C-terminal sequence of MT-3 is required for MT-3 to induce MET in HK-2 cells.ConclusionsThe results show that the HK-2 cell line can be an effective model to study later stages in the conversion of the renal epithelial cell to a mesenchymal cell. The HK-2 cell line, transfected with MT-3, may be an effective model to study the process of MET. The study implicates the unique C-terminal sequence of MT-3 in the conversion of HK-2 cells to display an enhanced epithelial phenotype.
Human skin is a well known target site of inorganic arsenic with effects ranging from hyperkeratosis to dermal malignancies. The current study characterizes the expression of a protein known to bind inorganic, As(3+), metallothionein 3 (MT-3). Expression of this protein was assessed immunohistochemically with a specific MT-3 antibody on human formalin-fixed, paraffin-embedded biopsy specimens in normal skin, squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and melanoma. Assessment in normal skin using nine normal specimens showed moderate to intense MT-3 staining in epidermal karatinocytes with staining extending into the basal cells and moderate to intense staining in melanocytes of nevi. MT-3 immunoexpression was shown to be moderate to intense in 12 of 13 of SCC, low to moderate in 8 of 10 BCC, and moderate to intense in 12 melanoma samples. MT-3 expression in cell culture models (normal human epidermal keratinocytes, normal human melanocytes, and HaCaT cells) showed only trace expression of MT-3, while exposures to the histone deacytalase inhibitor, MS-275, partially restored expression levels. These results indicate that the epidermis of human skin and resulting malignancies express high level of MT-3 and potentially impact on the known association of arsenic exposure and the development of skin disorders and related cancers.
Rho associated kinase p160 ROCK (Rock) and Ribosome S‐6 Kinase (RSK) both phosphorylate the Na+/H+ exchanger isoform 1 (NHE1) regulating ion transport, cell migration and binding to cytoskeletal linker proteins, ezrin, radixin, and moesin (ERM). NHE1 is an integral membrane protein involved in the regulation of intracellular pH, extracellular acidification, cellular migration, and cytoskeletal organization. Phosphorylation of NHE1 induces an intracellular alkalynization which promotes cellular proliferation and coordinates cellular migration through ERM binding and the accompanying organization of actin filaments. Using the Rock consensus sequence of RXXS/T and RXS/T and MS analysis of Rock phosphorylated NHE1, we have identified potential phosphorylation sites and created a series of site directed mutants. With the phosphorylation sites identified we have confirmed phosphorylation using in vitro labeling and generated stable cell lines expressing single and double Ser‐Ala mutations of the Rock and RSK sites to study changes in NHE regulation of ion translocation and cell motility events. This study intends to identify the Rock phosphorylation site on NHE1 and provide a new understanding for how transporters function to direct cell motility. This work was supported with funds from NSF‐MCB‐081778, NSF‐RUI‐MCB 0930432, and NIH‐1‐R15‐CA135616‐01
We previously reported a β‐adrenergic receptor (βAR) modulation of apoptosis requiring formation of a β‐arrestin/heat shock protein (HSP) 27 signaling complex for protection against program cell death. Our hypothesis is that a protective mechanism for this signalosome occurs by localized cellular trafficking of specific proteins. Non‐biased gene microarray profiling was used in epithelial (UROtsa) cells to identify gene products altered after co‐incubation with isoproterenol (ISO) and an apoptotic inducer, staurosporine (STS). Gene products found significantly changed when compared to STS alone included PRX, a mitochondrial peroxidase, FBXO28, a nuclear ubiquitin ligase and ZNF638, a pro‐apoptotic transcription factor. Quantitative real‐time PCR was used to validate changes of these gene products. There was increased transcription significantly different from STS only for PRX and FBXO in cells treated with ISO plus STS. Similar results were found for PRX using a neuronal cell model. Conversely, there was a significant decrease in ZNF638 transcription for UROtsa cells treated with ISO plus STS when compared to STS alone. All results corroborate observations from the gene microarray analysis. Higher PRX expression seen with concurrent ISO incubation is hypothesized as a compensatory mechanism for the increased O2− production initiated by STS. Supported by a SURF from ASPET and NIH grant P20RR016741.
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