Modulation of mitochondrial function by extracellular acidosis in tumor cells and normal fibroblasts: Role of signaling pathways.

In many tumors pronounced extracellular acidosis resulting from glycolytic metabolism is found. Since several environmental stress factors affect the mitochondrial activity the aim of the study was to analyze the impact of acidosis on cellular oxygen consumption and which signaling pathways may be involved in the regulation. In two tumor cell lines and normal fibroblasts cellular oxygen consumption rate (OCR) and mitochondrial function were measured after 3 h at pH 6.6. Besides the activation of ERK1/2, p38 and PI3K signaling in the cytosolic and mitochondrial compartment, the mitochondrial structure and proteins related to mitochondria fission were analyzed. The acidic extracellular environment increased OCR in tumor cells but not in fibroblasts. In parallel, the mitochondrial membrane potential increased at low pH. In both tumor lines (but not in fibroblasts), the phosphorylation of ERK1/2 and PI3K/Akt was significantly increased, and both cascades were involved in OCR modulation. The activation of signaling pathways was located predominantly in the mitochondrial compartment of the cells. At low pH, the mitochondrial structure in tumor cells showed structural changes related to elongation whereas mitochondria fragmentation was reduced indicating mitochondria fusion. However, these morphological changes were not related to ERK1/2 or PI3K signaling. Acidic stress seems to induce an increased oxygen consumption, which might further aggravate tumor hypoxia. Low pH also induces mitochondria fusion that is not mediated by ERK1/2 or PI3K signaling. The mechanism by which these signaling cascades modulate the respiratory activity of tumor cells needs further investigation.

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.

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.

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.

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.

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.

Endocytotic uptake of HPMA-based polymers by different cancer cells: impact of extracellular acidosis and hypoxia.

BACKGROUND: Polymeric nanoparticles allow to selectively transport chemotherapeutic drugs to the tumor tissue. These nanocarriers have to be taken up into the cells to release the drug. In addition, tumors often show pathological metabolic characteristics (hypoxia and acidosis) which might affect the polymer endocytosis. MATERIALS AND METHODS: Six different N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer structures (homopolymer as well as random and block copolymers with lauryl methacrylate containing hydrophobic side chains) varying in molecular weight and size were analyzed in two different tumor models. The cellular uptake of fluorescence-labeled polymers was measured under hypoxic (pO2 ≈1.5 mmHg) and acidic (pH 6.6) conditions. By using specific inhibitors, different endocytotic routes (macropinocytosis and clathrin-mediated, dynamin-dependent, cholesterol-dependent endocytosis) were analyzed separately. RESULTS: The current results revealed that the polymer uptake depends on the molecular structure, molecular weight and tumor line used. In AT1 cells, the uptake of random copolymer was five times stronger than the homopolymer, whereas in Walker-256 cells, the uptake of all polymers was much stronger, but this was independent of the molecular structure and size. Acidosis increased the uptake of random copolymer in AT1 cells but reduced the intracellular accumulation of homopolymer and block copolymer. Hypoxia reduced the uptake of all polymers in Walker-256 cells. Hydrophilic polymers (homopolymer and block copolymer) were taken up by all endocytotic routes studied, whereas the more lipophilic random copolymer seemed to be taken up preferentially by cholesterol- and dynamin-dependent endocytosis. CONCLUSION: The study indicates that numerous parameters of the polymer (structure, size) and of the tumor (perfusion, vascular permeability, pH, pO2) modulate drug delivery, which makes it difficult to select the appropriate polymer for the individual patient.

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.