Effect of Acidosis-Induced Signalling Pathways on Mitochondrial O2 Consumption of Tumour Cells.

Signalling pathways such as ERK1/2, p38 or PI3K are activated in tumour cells by extracellular acidosis, which is a common phenomenon in human tumours. These signalling pathways can modulate the mitochondrial function and activity. The aim of the study was to evaluate the impact of extracellular acidosis on the mitochondrial O2 consumption and, in consequence, the potential role of ERK1/2, p38 and PI3K cascades on modulating the respiratory function. The O2 consumption rate (OCR) was measured at pH 7.4 and extracellular acidosis (pH 6.6) in combination with inhibition of the respective signalling pathway. The activity of the pathways was determined by phosphorylation-specific western blot using the cytosolic and mitochondrial fraction of cell lysates. The experiments were performed on a rat tumour cell line (subline AT1 of the rat R-3327 prostate carcinoma) and normal cells (NRK-49F fibroblasts). Acidosis increased the OCR of AT1 cells, especially the basal OCR and the O2 consumption, which is related to ATP production. In normal NRKF cells OCR was unaffected by low pH. Inhibition of ERK1/2 and PI3K, but not p38, reduced the acidosis-induced increase of the OCR significantly in AT1 tumour cells. In this cell line acidosis also led to an ERK1/2 and PI3K activation, predominantly in the mitochondrial fraction. These results indicate that extracellular acidosis activates cellular respiration in tumour cells, presumably by activating the ERK1/2 and/or the PI3K signalling cascade. This activation of ERK1/2 and PI3K is located primarily in the mitochondrial compartment of the cells.

Acidosis-Induced Regulation of Egr1 and Ccn1 In Vitro and in Experimental Tumours In Vivo.

Extracellular acidosis is a characteristic of solid tumours, resulting from hypoxia-induced glycolytic metabolism as well as from the "Warburg effect" (aerobic glycolysis). The acidic environment has shown to affect functional tumour properties (proliferation, migration, invasion) and thus the aim of the study was to identify signalling mechanisms, mediating these pH-dependent effects. Therefore, the serum response factor (Srf) and the activation of the serum response element (SRE) by acidosis were analysed in AT-1 prostate carcinoma cells. Furthermore, the expression of downstream targets of this cascade, namely the early growth response 1 (Egr1), which seems to be involved in tumour proliferation, and the cellular communication network factor 1 (Ccn1), which both contain SRE in their promotor region were examined in two tumour cell lines. Extracellular acidification led to an upregulation of Srf and a functional activation of the SRE. Egr1 expression was increased by acidosis in AT-1 cells whereas hypoxia had a suppressive effect. In experimental tumours, in vivo Egr1 and Ccn1 were also found to be acidosis-dependent. Also, it turned out that pH regulated expression of Egr1 was followed by comparable changes of p21, which is an important regulator of the cell cycle.This study identifies the Srf-SRE signalling cascade and downstream Egr1 and Ccn1 to be acidosis-regulated in vitro and in vivo, potentially affecting tumour progression. Especially linked expression changes of Egr1 and p21 may mediate acidosis-induced effects on cell proliferation.

Role of the mTOR Signalling Pathway During Extracellular Acidosis in Tumour Cells.

The metabolic microenvironment of solid tumours is often dominated by extracellular acidosis which results from glycolytic metabolism. Acidosis can modulate gene expression and foster the malignant progression. The aim of the study was to analyse the effects of extracellular acidosis on the mTOR signalling pathway, an important regulator of anabolic and catabolic processes like cell proliferation and autophagy. The study was performed in two tumour cell lines, AT-1 prostate and Walker-256 mammary carcinoma cells. Cells were incubated at pH 7.4 or 6.6 for 3 h and 24 h. Then RNA and protein were extracted and analysed by qPCR and western blot. mTOR and P70-S6 kinase (P70-S6K), an important downstream target of mTOR, as well as the autophagic flux were studied. The effect of acidosis on P70S6K phosphorylation was compared to pharmacological mTOR inhibition with LY294002 and rapamycin. In both cell lines the total mTOR expression was not altered by acidosis, however, the mTOR phosphorylation was reduced after 3 h but not after 24 h. The P70S6K phosphorylation was reduced at both time points comparable to changes by pharmacological mTOR inhibitors. The autophagic flux, also a target of mTOR and measured by LC3-II expression, was increased in both cell lines after 24 h of acidosis. The results of this study indicate that mTOR signalling is inhibited by extracellular acidosis which then lead to a reduced activity of the P70-S6 kinase (modulating gene expression) and increased autophagy possibly mediated by ULK1/2 activity. These finding may offer new perspectives for therapeutic interventions in acidic tumours.

Impact of Acidosis-Regulated MicroRNAs on the Expression of Their Target Genes in Experimental Tumors In Vivo.

In comparison to normal tissue, solid tumors show an acidic extracellular pH, which results from hypoxia-induced glycolytic metabolism and the Warburg effect. Since acidosis modulates the expression of different microRNAs (e.g., miR-7, miR-183, miR-203, miR-215), microRNAs and their targets might be mediators between tumor acidosis and malignant behavior. The aim of this study was to investigate how modulation of these microRNAs affects the expression of their targets (Crem, cAMP-responsive element modulator; Gls2, glutaminase 2; Txnip, thioredoxin-interacting protein) in experimental tumors in vivo and whether these changes are acidosis dependent. The study was performed in two experimental tumor lines of the rat (AT-1 prostate carcinoma, Walker-256 mammary carcinoma). The results showed that all three targets were regulated by acidosis in vivo, Crem and Gls2 being downregulated and Txnip upregulated in both models. In AT-1 tumors at normal tumor pH, miR-203 overexpression increased Txnip expression by about 75%, whereas in Walker-256 tumors, miR-7 reduced protein expression. In more acidic tumors, no impact of microRNAs on Txnip expression was seen. On the other hand, Gls2 was significantly increased in acidic tumors by miR-183 or miR-7 overexpression (cell line dependent). As this increase was not present under control conditions, an acidosis-dependent effect can be assumed. These results indicate that tumor acidosis modulates the expression of targets of pH-sensitive microRNAs in experimental tumors. Especially the protein expression of Gls2 might be regulated via changes of microRNAs, which then affects the malignant progression of tumors.

The Acidic Tumor Microenvironment Affects Epithelial-Mesenchymal Transition Markers as Well as Adhesion of NCI-H358 Lung Cancer Cells.

Epithelial-mesenchymal transition (EMT), which is involved in metastasis formation, requires reprogramming of gene expression mediated by key EMT transcription factors. However, signals from the cellular microenvironment, including hypoxia, can also modulate the process of EMT. Hypoxia is often associated with a reduction in the extracellular pH of the tumor microenvironment (acidosis). Whether acidosis alone has an impact on the expression of the EMT markers E-cadherin, N-cadherin, and vimentin was studied in NCI-H358 lung cancer cells. Reducing extracellular pH decreased E-cadherin mRNA, while vimentin and N-cadherin mRNA were doubled. However, at the protein level, E-cadherin and N-cadherin were both reduced, and only vimentin was upregulated. E-cadherin and N-cadherin expression at the cell surface, which is the relevant parameter for cell-cell and cell-matrix interaction, decreased too. The reduction of cell surface proteins was due to diminished protein expression and not changes in cellular localization, since localization of EMT markers in general was not affected by acidosis. Acidosis also affected NCI-H358 cells functionally. Adhesion was decreased when the cells were primed in an acidic medium before measuring cell adherence, which is in line with the reduced expression of cadherins at the cell surface. Additionally, migration was decreased after acidic priming. A possible mechanism for the regulation of EMT markers involves the action of microRNA-203a (miR-203a). In NCI-H358 lung cancer cells, miR-203a expression was repressed by acidosis. Since a decrease in the level of miR-203a has been shown to induce EMT, it might be involved in the modulation of EMT marker expression, adhesion, and migration by the acidic tumor microenvironment in NCI-H358 lung cancer cells.

Extracellular Acidosis Regulates the Expression of Inflammatory Mediators in Rat Epithelial Cells.

Acidification of the cellular microenvironment is found in different pathological states such as inflammation, ischemia and in solid tumors. It can affect cell function and phenotype, and by this aggravate the pathological process. Epithelial cells are a relevant functional part in several normal organs as well as in tumors and will thus be challenged by the acidic extracellular pH (acidosis). Therefore, the impact of acidosis on the expression of different inflammatory mediators (MCP-1, IL-6, osteopontin, iNOS, TNF-α, and COX-2), as well as the role of different signaling pathways regulating the expression, was studied in epithelial normal rat kidney cells (NRK-52E). Acidosis led to an increase in TNF-α expression but a down-regulation of MCP-1, iNOS and COX-2. Expression of IL-6 was only slightly modulated, while osteopontin was not regulated at all. Since acidosis activates ERK1/2 and p38 signaling in NRK-52E cells, the impact of MAP kinase signaling pathways on the expression of the inflammatory markers was analyzed. At normal pH, blocking ERK1/2 or p38 decreased the level of MCP-1, iNOS and partly TNF-α. However, the effect of acidosis on the expression of inflammatory mediators was not affected by inhibition of the MAP kinase pathways. In conclusion, our results show that an acidic microenvironment affects the transcriptional program of epithelial cells. Low pH mostly reduced the expression of pathological relevant genes and might thus repress inflammatory processes induced by epithelial cells.

Expression of MicroRNAs in Fibroblasts and Macrophages Is Regulated by Hypoxia-Induced Extracellular Acidosis.

Under pathological conditions like inflammation, ischemia or in solid tumors, parameters of the microenvironment like local oxygenation and extracellular pH show marked changes when compared to healthy tissue. The altered microenvironment affects cellular phenotype of omnipresent fibroblasts and immune cells. Recently, the impact of the microenvironment on the expression patterns of microRNAs, small non-coding RNAs that regulate gene expression on a post-transcriptional level, was discussed. Therefore, microRNAs might be the link between altered microenvironmental parameters and changes in cellular phenotype. In this study, the effect of hypoxia-induced extracellular acidosis (24 h pH 6.6) on microRNA expression in fibroblasts and macrophages was analyzed. MicroRNAs in rat fibroblasts (NRK-49F) were examined with the miScript miRNA PCR Array and changes in the expression validated by TaqMan qPCR. Subsequently, the identified microRNAs were analyzed in RAW 264.7 mouse macrophages. Nine out of 84 tested microRNAs were found to be acidosis-regulated in fibroblasts by miRNA PCR array, most of them up-regulated. Of those, the pH dependency could be validated by TaqMan qPCR for five of these nine microRNAs. When comparing these microRNAs in terms of their expression in macrophages, profound differences were observed. Thus, acidosis-induced alterations in the expression of microRNAs seem to be cell-type specific. Only the up-regulation of the miR-133b by low pH was seen in all normal cells, but not in tumor cells. As the identified microRNAs are involved in the regulation of proliferation, cell death and migration (amongst others), acidosis-induced changes in their expression might affect cellular behavior of fibroblasts and macrophages under pathological conditions. For instance the proto-oncogene c-Jun, which is a target of the miR-133b, was shown to be acidosis-regulated. Acidosis could regulate the biological behavior via miRNA-133b and c-Jun.

Hypoxia-Related Tumor Acidosis Affects MicroRNA Expression Pattern in Prostate and Breast Tumor Cells.

MicroRNAs (miRNAs) are small non-coding RNA sequences which are able to modulate the expression of many functional proteins. The expression level of miRNAs can be modulated by parameters of the tumor microenvironment like hypoxia, nutrient deprivation or oxidative stress. Since miRNAs can act either as oncogenes or tumor suppressors, this may affect malignant progression or therapy resistance. In the present study it was analyzed whether extracellular acidosis can impact on miRNA expression. Therefore, tumor cells (R3327-AT-1 prostate and Walker-256 mammary carcinoma cells) were incubated at pH 6.6 (acidosis) or pH 7.4 (control) for 24 h and changes in miRNA expression were analyzed by PCR array for 84 cancer-associated miRNAs and Next-Generation Sequencing (NGS) with a panel of 765 miRNAs.In the cancer-related PCR array an acidosis-induced reduction of 5 miRNAs in AT-1 and 6 miRNAs in Walker-256 cells was seen. The miR-203a was consensually down-regulated in both cell lines. Using NGS, 19 miRNAs were found to be upregulated and 14 miRNAS were downregulated in AT-1 prostate cancer cells. In Walker-256 cells the expression of 21 miRNAs was increased and decreased for 17 miRNAs. Eleven miRNAs were regulated by acidosis in both tumor cell lines in the same direction.Acidosis induced changes in the miRNA expression of prostate and breast carcinoma cells. However, miRNA profiles differed strongly between the tumor cell lines (and between the experimental methods used), indicating that cells can react individually to microenvironmental stress. However, some miRNAs were consensually regulated in both cell lines and thus might represent a general cellular response to an extracellular acidosis.

Impact of the Tumor Microenvironment on the Expression of Inflammatory Mediators in Cancer Cells.

Hypoxia and extracellular acidosis are common features of solid malignant tumors. The aim of the study was to analyze whether these pathophysiological parameters affect the expression of inflammatory mediators in tumor cells. Therefore the mRNA expression of MCP-1 (monocyte chemotactic protein 1), iNOS and osteopontin was measured under hypoxic (pO2 1 mmHg) and acidotic (pH 6.6) conditions by qPCR in AT1 R-3327 prostate cancer cells. In addition, the underlying signaling cascades were analyzed by using inhibitors of the p38 and ERK1/2 MAP kinase pathways.Hypoxia led to a significant decrease of the expression of MCP-1 and osteopontin over the complete observation period of 24 h, whereas the iNOS expression after an initial reduction slightly increased. Acidotic conditions for up to 6 h increased the iNOS expression significantly which was functional as indicated by an elevated level of nitrate/nitrite formation by 30 %. Acidosis had almost no impact on the MCP-1 expression of tumor cells, whereas the osteopontin level tended to increase leading to a significantly elevated level after 24 h at pH 6.6. Inhibiting the p38 and ERK1/2 under control conditions revealed that the MAPKs play a significant role for the regulation of the expression of inflammatory mediators. MCP-1 expression could be lowered by inhibiting ERK1/2 whereas iNOS expression was dependent on both p38 and ERK1/2 MAPK. These results indicate that the adverse tumor microenvironment affects the expression of inflammatory mediators by tumors cells and may therefore modulate the immune response within the tumor tissue.

Contributions from lateral interaction mechanisms to the human ERG can be studied with a two-frequency method.

We present a two-frequency method to investigate lateral interaction components (liERGs) in the human electroretinogram. Adjacent half cycles of sinusoidal gratings were modulated sinusoidally with different temporal frequencies f1 and f2. The liERGs were defined by the Fourier components at the intermodulation frequencies /f1 - f2/ and f1 + f2 which indicate nonlinear interactions between the half cycles. Significant liERGs were found in all subjects with a monotonic increase of the liERG magnitude in the spatial frequency range from f(s)=0.07 to 2.4 cpd. When /f1 - f2/ was below 5 Hz, liERGs were masked by noise intrusions. In a control experiment we demonstrated that the liERGs were not evoked by stray light artifacts. The liERGs may help to further differentiate the responses that are evoked by patterned stimuli within the retina.