The Role of microRNAs in Gene Expression and Signaling Response of Tumor Cells to an Acidic Environment.

Many tumors are characterized by marked extracellular acidosis due to increased glycolytic metabolism, which affects gene expression and thereby tumor biological behavior. At the same time, acidosis leads to altered expression of several microRNAs (Mir7, Mir183, Mir203, Mir215). The aim of this study was to analyze whether the acidosis-induced changes in cytokines and tumor-related genes are mediated via pH-sensitive microRNAs. Therefore, the expression of Il6, Nos2, Ccl2, Spp1, Tnf, Acat2, Aox1, Crem, Gls2, Per3, Pink1, Txnip, and Ypel3 was examined in acidosis upon simultaneous transfection with microRNA mimics or antagomirs in two tumor lines in vitro and in vivo. In addition, it was investigated whether microRNA expression in acidosis is affected via known pH-sensitive signaling pathways (MAPK, PKC, PI3K), via ROS, or via altered intracellular Ca2+ concentration. pH-dependent microRNAs were shown to play only a minor role in modulating gene expression. Individual genes (e.g., Ccl2, Txnip, Ypel3) appear to be affected by Mir183, Mir203, or Mir215 in acidosis, but these effects are cell line-specific. When examining whether acid-dependent signaling affects microRNA expression, it was found that Mir203 was modulated by MAPK and ROS, Mir7 was affected by PKC, and Mir215 was dependent on the intracellular Ca2+ concentration. Mir183 could be increased by ROS scavenging. These correlations could possibly result in new therapeutic approaches for acidotic tumors.

Correlation between Circulating miR-16, miR-29a, miR-144 and miR-150, and the Radiotherapy Response and Survival of Non-Small-Cell Lung Cancer Patients.

Despite the success of current therapy concepts, patients with advanced non-small-cell lung cancer (NSCLC) still have a very poor prognosis. Therefore, biological markers are urgently needed, which allow the assessment of prognosis, or prediction of the success of therapy or resistance in this disease. Circulating microRNAs (miRs) have potential as biomarkers for the prognosis and prediction of response to therapy in cancer patients. Based on recent evidence that circulating miR-16, miR-29a, miR-144 and miR-150 can be regulated by ionizing radiation, the concentration of these four miRs was assessed in the plasma of NSCLC patients at different time points of radiotherapy by digital droplet PCR (ddPCR). Furthermore, their impact on patients' prognosis was evaluated. The mean plasma levels of miR-16, miR-29a, miR-144 and miR-150 significantly differed intra- and inter-individually, and during therapy in NSCLC patients, but showed a strong positive correlation. The individual plasma levels of miR-16, miR-29a and miR-144 had prognostic value in NSCLC patients during or at the end of radiotherapy in Cox's regression models. NSCLC patients with low levels of these three miRs at the end of radiotherapy had the worst prognosis. However, miR-150 plasma levels and treatment-dependent changes were not predictive. In conclusion, circulating miR-16, miR-29a and miR-144, but not miR-150, have a prognostic value in NSCLC patients undergoing radiotherapy.

Role of acidosis-sensitive microRNAs in gene expression and functional parameters of tumors in vitro and in vivo.

BACKGROUND: The acidic extracellular environment of tumors has been shown to affect the malignant progression of tumor cells by modulating proliferation, cell death or metastatic potential. The aim of the study was to analyze whether acidosis-dependent miRNAs play a role in the signaling cascade from low pH through changes in gene expression to functional properties of tumors in vitro and in vivo. METHODS: In two experimental tumor lines the expression of 13 genes was tested under acidic conditions in combination with overexpression or downregulation of 4 pH-sensitive miRNAs (miR-7, 183, 203, 215). Additionally, the impact on proliferation, cell cycle distribution, apoptosis, necrosis, migration and cell adhesion were measured. RESULTS: Most of the genes showed a pH-dependent expression, but only a few of them were additionally regulated by miRNAs in vitro (Brip1, Clspn, Rif1) or in vivo (Fstl, Tlr5, Txnip). Especially miR-215 overexpression was able to counteract the acidosis effect in some genes. The impact on proliferation was cell line-dependent and most pronounced with overexpression of miR-183 and miR-203, whereas apoptosis and necrosis were pH-dependent but not influenced by miRNAs. The tumor growth was markedly regulated by miR-183 and miR-7. In addition, acidosis had a strong effect on cell adhesion, which could be modulated by miR-7, miR-203 and miR-215. CONCLUSIONS: The results indicate that the acidosis effect on gene expression and functional properties of tumor cells could be mediated by pH-dependent miRNAs. Many effects were cell line dependent and therefore do not reflect universal intracellular signaling cascades. However, the role of miRNAs in the adaptation to an acidic environment may open new therapeutic strategies.

The Role of MicroRNA Expression for Proliferation and Apoptosis of Tumor Cells: Impact of Hypoxia-Related Acidosis.

The metabolic microenvironment in tumors is characterized by hypoxia and acidosis. Extracellular pH sometimes decreases to even below 6.0. Previous experiments showed that tissue pH has an impact on tumor cell proliferation and apoptosis. However, the mechanism of how cell cycle progression is affected by decreased pH is not fully understood yet. One possible mechanism includes changes in the expression of miRNAs. The aim of this study was to analyze the impact of pH-regulated miRNAs (miR-183 and miR-215) on proliferation, apoptosis, and necrosis of tumor cells. Therefore, AT1 prostate and Walker-256 mammary carcinoma cells were transfected with the miRNAs or with the respective antagomirs and incubated at pH 7.4 and 6.6 for 24 h. AT1 cells underwent a G0/G1 cell cycle arrest under acidic conditions and showed a marked reduction of the number of actively DNA-synthesizing cells. In Walker-256 cells, acidosis induced a reduction of apoptosis and additionally a significant increase in necrotic cell death. Transfection of tumor cells with miR-183 or miR-215, which were significantly downregulated under acidic conditions, had no impact on cell death of AT1 or Walker-256 cells. Overexpression of miR-183, which is also downregulated by acidosis, intensified G0/G1 cell cycle arrest in AT1 cells. Previous studies revealed that hypoxia-related tumor acidosis affects the expression of different small noncoding RNAs. However, not all of these acidosis-regulated miRNAs seem to have an impact on proliferation, apoptosis, and necrosis of tumor cells. While miR-215 had no influence, miR-183 seems to be an interesting candidate that could amplify the impact of extracellular acidosis on malignant behavior of tumor cells.

Functional Impact of Acidosis-Regulated MicroRNAs on the Migration and Adhesion of Tumor Cells.

Tumor tissue shows special features in metabolism in contrast to healthy tissue. Besides a distinctive oxygen deficiency, tumors often show a reduced extracellular pH (acidosis) resulting from an intensified glycolysis not only under hypoxic but also under normoxic conditions (Warburg effect). As shown in previous studies, cell migration is increased in AT1 prostate carcinoma cells after incubation at pH 6.6, and this leads to an increased number of lung metastases in vivo. However, the signaling pathway causing these functional changes is still unknown. Possible mediators could be acidosis-regulated microRNAs (miR-7, miR-183, miR-203, miR-215). The aim of the study was therefore to analyze whether a change in the expression of these microRNAs has an impact on the tumor cell migration and adhesion. Studies were performed with AT1 rat prostate cancer cells which were incubated for 24 h at pH 7.4 or 6.6. Keeping AT1 tumor cells at low pH increased the migratory capacity by about 100%. But also the decrease of miR-203 and miR-215 expression (at normal pH) led to an increase in migration velocity by 50%. In contrast, cell adhesion was increased by about 75% at low pH. However, an increase in miR-215 expression at pH 6.6 reduced the adhesion by trend. These results clearly indicated that the extracellular pH has an impact on migration and adhesion of tumor cells. In this mechanism, pH-regulated microRNAs could play a role since changes in the expression of these microRNAs (especially miR-203) are also able to modulate the migratory behavior.

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.

Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo.

BACKGROUND: The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. METHODS: Gene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed. RESULTS: NGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH. CONCLUSIONS: These data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

Causes and Consequences of A Glutamine Induced Normoxic HIF1 Activity for the Tumor Metabolism.

The transcription factor hypoxia-inducible factor 1 (HIF1) is the crucial regulator of genes that are involved in metabolism under hypoxic conditions, but information regarding the transcriptional activity of HIF1 in normoxic metabolism is limited. Different tumor cells were treated under normoxic and hypoxic conditions with various drugs that affect cellular metabolism. HIF1α was silenced by siRNA in normoxic/hypoxic tumor cells, before RNA sequencing and bioinformatics analyses were performed while using the breast cancer cell line MDA-MB-231 as a model. Differentially expressed genes were further analyzed and validated by qPCR, while the activity of the metabolites was determined by enzyme assays. Under normoxic conditions, HIF1 activity was significantly increased by (i) glutamine metabolism, which was associated with the release of ammonium, and it was decreased by (ii) acetylation via acetyl CoA synthetase (ACSS2) or ATP citrate lyase (ACLY), respectively, and (iii) the presence of L-ascorbic acid, citrate, or acetyl-CoA. Interestingly, acetylsalicylic acid, ibuprofen, L-ascorbic acid, and citrate each significantly destabilized HIF1α only under normoxia. The results from the deep sequence analyses indicated that, in HIF1-siRNA silenced MDA-MB-231 cells, 231 genes under normoxia and 1384 genes under hypoxia were transcriptionally significant deregulated in a HIF1-dependent manner. Focusing on glycolysis genes, it was confirmed that HIF1 significantly regulated six normoxic and 16 hypoxic glycolysis-associated gene transcripts. However, the results from the targeted metabolome analyses revealed that HIF1 activity affected neither the consumption of glucose nor the release of ammonium or lactate; however, it significantly inhibited the release of the amino acid alanine. This study comprehensively investigated, for the first time, how normoxic HIF1 is stabilized, and it analyzed the possible function of normoxic HIF1 in the transcriptome and metabolic processes of tumor cells in a breast cancer cell model. Furthermore, these data imply that HIF1 compensates for the metabolic outcomes of glutaminolysis and, subsequently, the Warburg effect might be a direct consequence of the altered amino acid metabolism in tumor cells.

Correction to: Tumor pH and metastasis: a malignant process beyond hypoxia.

The authors have noticed a typographical error in the published article. The term "epithelial-to-mesenchymal transition" should have been used instead of the term "endothelial-to-mesenchymal transition" throughout the manuscript.

Extracellular Acidosis Modulates the Expression of Epithelial-Mesenchymal Transition (EMT) Markers and Adhesion of Epithelial and Tumor Cells.

Epithelial-to-mesenchymal transition (EMT) is an important process of tumor progression associated with increased metastatic potential. EMT can be activated by external triggers such as cytokines or metabolic parameters (e.g. hypoxia). Since extracellular acidosis is a common finding in tumors, the aim of the study is to analyze its impact on the expression of EMT markers in vitro and in vivo as well as the functional impact on cell adhesion. Therefore, three tumor and two normal epithelial cell lines were incubated for 24 h at pH 6.6 and the expression of EMT markers was studied. In addition, mRNA expression of transcription and metabolic factors related to EMT was measured as well as the functional impact on cell adhesion, either during acidic incubation or after priming cells in an acidic milieu. E-cadherin and N-cadherin were down-regulated in all tumor and normal cell lines studied, whereas vimentin expression increased in only two tumor and one normal cell line. Down-regulation of the cadherins was seen in total protein and to a lesser extent in surface protein. In vivo an increase in N-cadherin and vimentin expression was found. Acidosis up-regulated Twist1 and Acsl1 but down-regulated fumarate hydratase (Fh). Cell adhesion during acidic incubation decreased in AT1 prostate carcinoma cells whereas preceding acidic priming increased their subsequent adhesion. Low tumor pH is able to modulate the expression EMT-related proteins and by this may affect the stability of the tissue structure.

Cellular and radiobiological effects of carbonic anhydrase IX in human breast cancer cells.

Hypoxia­induced carbonic anhydrase IX (CAIX) is involved in intracellular and extracellular pH regulation, which is critical for tumor growth and metastasis. CAIX is overexpressed in breast cancer and is associated with the poor survival of patients after radiotherapy. Therefore, we evaluated the cellular and radiobiological effects of CAIX inhibition in human breast cancer cells. We used CA9 siRNA and the CA inhibitor (CAI) U104, respectively, to inhibit CAIX expression and activity in basal triple­negative MDA­MB­231 and luminal MCF­7 cells under hypoxic conditions. We investigated the effects of CAIX inhibition on CA9 mRNA and CAIX protein level, as well as on CAIX activity, intracellular pH, proliferation, apoptosis, clonogenic survival, migration, cell cycle distribution and radiosensitivity. CA9 siRNA and CAI U104 decreased CA9 mRNA and CAIX protein level in MDA­MB­231 and MCF­7 cells. Furthermore, incubation with CAI U104 significantly decreased carbonic anhydrase activity and reduced the intracellular pH. Additionally, CA9 siRNA or U104 reduced clonogenic survival, migration and the number of cells in the G0/G1 phase, induced apoptosis and demonstrated additive or synergistic effects in combination with irradiation. In conclusion, combination of CAIX inhibition and irradiation is a promising treatment strategy against breast cancer with hypoxia­induced CAIX expression.

Tumor pH and metastasis: a malignant process beyond hypoxia.

Tumors often show, compared to normal tissues, a markedly decreased extracellular pH resulting from anaerobic or aerobic glycolysis in combination with a reduced removal of acidic metabolites. Several studies indicate that acidosis induces (independently from hypoxia) hematogenous and lymphatic spread of tumor cells worsening the long-term prognosis of tumor patients. This review gives an overview on the impact of low pH on different steps of metastasis including (a) local tumor cell invasion and angiogenesis, (b) intravasation of tumor cells and detachment into the circulation, and (c) adherence of circulating tumor cells, transmigration and invasion in the new host tissue. The review describes pH-dependent cellular mechanisms fostering these steps such as epithelial-to-mesenchymal transition (EMT), activation of cell migration, degradation of the extracellular matrix, or angiogenesis. The review discusses mechanisms of tumor cells for proton sensing including acid-sensitive ion channels (ASICs, TRPs) or ion transporters (NHE1) and G protein coupled H+-sensors. Finally, the review describes several intracellular signaling cascades activated by H+ sensing mechanisms leading to transcriptional, post-transcriptional, or functional changes in the cell relevant for the metastatic spread. From these studies, different therapeutical approaches are described to overcome tumor acidosis or to interfere with the signaling cascades to reduce the metastatic potential of tumors.

Acidic extracellular environment affects miRNA expression in tumors in vitro and in vivo.

Hypoxia can control the expression of miRNAs in tumors which play an important role for the control of the malignant behavior. The aim of the study was to analyze whether extracellular acidosis, a common feature of tumors, also has an impact on the miRNA expression in isolated cells as well as in solid tumors. MiRNA expression was analyzed in two rat tumor cell lines (AT1 prostate and Walker-256 mammary carcinomas) by NGS and qPCR. In vivo the same cell lines were implanted subcutaneously and the tumor pH was modulated by inspiratory hypoxia and inhibition of the respiratory chain. In addition, the expression of five genes (Brip1, Ercc6l, Ikbke, Per3, Tlr5) which are potential targets of the miRNAs were analyzed on mRNA level. Screening showed that 38 (AT1) resp. 41 (Walker-256) miRNAs were pH-dependent. Validation by qPCR revealed that only 4 miRNAs were consistently regulated in both cell lines: miR-183, miR-203a, miR-215 and miR-7a. The expression of miR-7a was increased by low pH whereas all others were decreased. In the tumors in vivo all 4 miRNAs were down-regulated. The potential targets showed pH dependency in both cell lines. In conclusion, extracellular acidosis regulates the expression of miRNAs in vitro and in vivo. The expression of targets of these miRNAs were also pH-dependent. The pH may therefore affect the biological behavior of tumors via miRNAs.

Inhibition of Carbonic Anhydrase IX by Ureidosulfonamide Inhibitor U104 Reduces Prostate Cancer Cell Growth, But Does Not Modulate Daunorubicin or Cisplatin Cytotoxicity.

Carbonic anhydrase (CA) IX has emerged as a promising target for cancer therapy. It is highly upregulated in hypoxic regions and mediates pH regulation critical for tumor cell survival as well as extracellular acidification of the tumor microenvironment, which promotes tumor aggressiveness via various mechanisms, such as augmenting metastatic potential. Therefore, the aim of this study was to analyze the complex interdependency between CA IX and the tumor microenvironment in prostate tumor cells with regard to potential therapeutic implications. CA IX was upregulated by hypoxia as well as acidosis in prostate cancer cells. This induction did not modulate intracellular pH but led to extracellular acidification. Pharmacological inhibition of CA IX activity by U104 (SLC-0111) resulted in a reduction in tumor cell growth and an increase in apoptotic cell death. Intracellular pH was reduced under normoxic and even more so under hypoxic conditions when CA IX level was high. However, although intracellular pH regulation was disturbed, targeting CA IX in combination with daunorubicin or cisplatin did not intensify apoptotic tumor cell death. Hence, targeting CA IX in prostate cancer cells can lead to intracellular pH dysregulation and, consequently, can reduce cellular growth and elevate apoptotic cell death. Attenuation of extracellular acidification by blocking CA IX might additionally impede tumor progression and metastasis. However, no beneficial effect was seen when targeting CA IX in combination with chemotherapeutic drugs.

Tumor Acidosis and Hypoxia Differently Modulate the Inflammatory Program: Measurements In Vitro and In Vivo.

Inflammatory mediators produced by the tumor cells are of importance for immune response but also for malignant progression. The aim of the study was to analyze the expression of monocyte chemoattractant protein-1, interleukin-6 (IL-6), tumor necrosis factor-α, inducible isoform of nitric oxide synthase (iNOS), cyclooxygenase-2, and osteopontin in vitro in two different tumor cell lines under hypoxia (pO2≈1.5 mmHg) and/or acidosis (pH=6.6) for up to 24 hours since hypoxia and acidosis are common characteristics of solid tumors. Additionally, the same tumor cell lines implanted in vivo were made hypoxic and acidotic artificially for 24 hours, after which the cytokine expression was measured. Finally, the activation of ERK1/2 and p38 by acidosis/hypoxia and their impact on cytokine expression were studied. The results indicate that acidosis and hypoxia have fundamentally different (often opposing) effects on cytokine expression. In addition, these effects were tumor cell line specific. When combining hypoxia and acidosis, the overall changes reflect an additive effect of both conditions alone, indicating that hypoxia and acidosis act by independent mechanisms. The in vivo changes corresponded well with the results obtained in the isolated tumor cells. Only iNOS expression was downregulated in vivo but increased in cell culture. For IL-6 expression, the acidosis-induced changes were dependent on ERK1/2 activation. In conclusion, it was demonstrated that the environmental pO2 and pH strongly affect the expression of inflammatory mediators in tumor cells. In vivo, most of the inflammatory mediators were downregulated, which could limit the activation of immune cells and by this foster the immune escape of tumors.

Acidosis differently modulates the inflammatory program in monocytes and macrophages.

Inflammation, ischemia or the microenvironment of solid tumors is often accompanied by a reduction of extracellular pH (acidosis) that stresses the cells and acts on cellular signaling and transcription. The effect of acidosis on the expression of various inflammatory markers, on functional parameters (migration, phagocytic activity) and on signaling pathways involved was studied in monocytic cells and macrophages. In monocytic cell lines acidosis led to a reduction in expression of most of the inflammatory mediators, namely IL-1ß, IL-6, TNF-α, MCP-1, COX-2 and osteopontin. In primary human monocytes MCP-1 and TNF-α were reduced but COX-2 and IL-6 were increased. In RAW264.7 macrophage cell line IL-1ß, COX-2 and iNOS expression was increased, whereas MCP-1 was reduced similar to the effect in monocytic cells. For primary human monocyte-derived macrophages the regulation of inflammatory markers by acidosis depended on activation state, except for the acidosis-induced downregulation of MCP-1 and TNF-α. Acidosis affected functional immune cell behavior when looking at phagocytic activity which was increased in a time-dependent manner, but cellular motility was not changed. Neither ERK1/2 nor CREB signaling was stimulated by the reduction of extracellular pH. However, p38 was activated by acidosis in RAW264.7 cells and this activation was critical for the induction of IL-1ß, COX-2 and iNOS expression. In conclusion, acidosis may impede the recruitment of immune cells, but fosters inflammation when macrophages are present by increasing the level of COX-2 and iNOS and by functionally forcing up the phagocytic activity.

Impact of hypoxia-related tumor acidosis on cytotoxicity of different chemotherapeutic drugs in vitro and in vivo.

Extracellular acidosis in tumors leads to an activation of the p-glycoprotein (Pgp) drug transporter. In the present study the cytotoxicity of different chemotherapeutic drugs and its dependence on the Pgp activity during acidosis were analyzed in vitro and in vivo. Treating R3327-AT1, Pgp-positive tumor cells at pH 7.4 with daunorubicin, cisplatin or docetaxel led to marked apoptosis induction and cell death. Under acidic (pH 6.6) conditions cytotoxicity of daunorubicin or docetaxel was significantly reduced whereas cisplatin-induced cell death was almost pH-independent. Inhibiting Pgp with verapamil reversed the acidosis-induced chemoresistance against daunorubicin and docetaxel. The Pgp expression was unaffected by pH. In vivo the cytotoxicity of daunorubicin and docetaxel was also pH dependent. When acidifying the tumors by forcing glycolytic metabolism, apoptosis induction decreased significantly indicating a reduced chemosensitivity. The cytotoxic effect of cisplatin in vivo was unaffected by the tumor pH. Since daunorubicin and docetaxel (but not cisplatin) are substrates of the Pgp, these results underline the influence of the tumor acidosis on the Pgp-mediated chemoresistance which can be counteracted by inhibition of the drug transporter.

Acidic priming enhances metastatic potential of cancer cells.

Metabolic acidosis is a common feature of tumor microenvironment and may affect the phenotype of tumor cells, including invasive capacity and formation of metastases. We tested whether previous exposure to an acidic environment alters metastatic potential of two rat carcinoma cell lines in the animal model. In addition, we determined the effect of an acidic environment on motility and invasive capacity of AT-1 prostate carcinoma cells in culture. Exposure of tumor cells to an acidic environment (pH 6.6, 5 % CO2, 6 h) prior to tail vein injection in rats enhanced formation of lung metastases significantly. In culture, acidosis increased cellular motility of AT-1 cells. When the tumor cells were transferred back to pH 7.4, enhanced motility persisted for at least 3 h but vanished after longer periods (24 h), therefore presenting a "short-term memory effect." Although acidosis augmented phosphorylation of ERK1/2 and p38, and inhibition of ERK1/2 phosphorylation or of p38 kinase activity reduced basal motility at pH 7.4, acidosis-induced increase in motility was not dependent on ERK1/2 or p38 kinase. Src family kinases were not involved either. By contrast, scavenging reactive oxygen species (ROS), known to be increased in AT-1 cells under acidic conditions, blunted acidosis-induced motility increase. Our data indicate that tumor cells may acquire enhanced motility in an acidic micromilieu, at least in part due to enhanced ROS formation. Because enhanced motility persists for at least 3 h after leaving the acidic environment, this may promote metastasis formation, as observed in our in vivo model.

Impact of extracellular acidosis on intracellular pH control and cell signaling in tumor cells.

Cells in solid tumors generate an extracellular acidosis due to the Warburg effect and tissue hypoxia. Acidosis can affect the functional behavior of tumor cells, causing, e.g., multidrug resistance. In this process ERK1/2 and p38 mitogen-activated protein kinases (MAPK) seem to play a key role. However, the underlying mechanism of MAPK activation by extracellular acidosis remains unclear. Experiments were performed in three tumor and three normal tissue cell lines in which the cells were exposed to an extracellular pH of 6.6 for 3 h. Intracellular pH (pHi), protein expression and activation, acidosis-induced transactivation, and reactive oxygen species (ROS) formation were measured. Extracellular acidosis resulted in a rapid and sustained decrease of pHi leading to a reversal of the extra-/intracellular pH gradient. Extracellular acidosis led to p38 phosphorylation in all cell types and to ERK1/2 phosphorylation in three of six cell lines. Furthermore, p38 phosphorylation was also observed during sole intracellular lactacidosis at normal pHe. Acidosis-enhanced formation of ROS, probably originating from mitochondria, seems to trigger MAPK phosphorylation. Finally, acidosis increased phosphorylation of the transcription factor CREB and resulted in increased transcriptional activity. Thus, an acidic tumor microenvironment can induce a longer-lasting p38 CREB-mediated change in the transcriptional program.

Acidic environment leads to ROS-induced MAPK signaling in cancer cells.

Tumor micromilieu often shows pronounced acidosis forcing cells to adapt their phenotype towards enhanced tumorigenesis induced by altered cellular signalling and transcriptional regulation. In the presents study mechanisms and potential consequences of the crosstalk between extra- and intracellular pH (pH(e), pH(i)) and mitogen-activated-protein-kinases (ERK1/2, p38) was analyzed. Data were obtained mainly in AT1 R-3327 prostate carcinoma cells, but the principle importance was confirmed in 5 other cell types. Extracellular acidosis leads to a rapid and sustained decrease of pH(i) in parallel to p38 phosphorylation in all cell types and to ERK1/2 phosphorylation in 3 of 6 cell types. Furthermore, p38 phosphorylation was elicited by sole intracellular lactacidosis at normal pH(e). Inhibition of ERK1/2 phosphorylation during acidosis led to necrotic cell death. No evidence for the involvement of the kinases c-SRC, PKC, PKA, PI3K or EGFR nor changes in cell volume in acidosis-induced MAPK activation was obtained. However, our data reveal that acidosis enhances the formation of reactive oxygen species (ROS), probably originating from mitochondria, which subsequently trigger MAPK phosphorylation. Scavenging of ROS prevented acidosis-induced MAPK phosphorylation whereas addition of H(2)O(2) enhanced it. Finally, acidosis increased phosphorylation of the transcription factor CREB via p38, leading to increased transcriptional activity of a CRE-reporter even 24 h after switching the cells back to a normal environmental milieu. Thus, an acidic tumor microenvironment can induce a longer lasting p38-CREB-medited change in the transcriptional program, which may maintain the altered phenotype even when the cells leave the tumor environment.