CA IX Stabilizes Intracellular pH to Maintain Metabolic Reprogramming and Proliferation in Hypoxia.

Tumor hypoxia represents a severe microenvironmental stress that is frequently associated with acidosis. Cancer cells respond to these stresses with changes in gene expression that promote survival at least in part through pH regulation and metabolic reprogramming. Hypoxia-induced carbonic anhydrase IX (CA IX) plays a critical adaptive role in response to hypoxic and acidic environments by catalytically hydrating extracellular CO2 to produce bicarbonate for buffering intracellular pH (pHi). We used proteome-wide profiling to study the cellular response to transient CA IX knockdown in hypoxia and found a decrease in the levels of key glycolytic enzymes and lactate dehydrogenase A (LDHA). Interestingly, the activity of LDH was also decreased as demonstrated by native in-gel activity assay. These changes led to a significant reduction in glycolytic flux and extracellular lactate levels in cancer cells in vitro, contributing to a decrease in proliferation. Interestingly, addition of the alternative LDH substrate alpha-ketobutyrate restored LDHA activity, extracellular acidification, pHi, and cellular proliferation. These results indicate that in the absence of CA IX, reduction of pHi disrupts LDHA activity and hinders the cellular capacity to regenerate NAD+ and secrete protons to the extracellular space. Hypoxia-induced CA IX therefore mediates adaptation to microenvironmental hypoxia and acidosis directly, by enzymatically converting extracellular CO2 to bicarbonate, and indirectly, by maintaining glycolysis-permissive intracellular milieu.

CAIX-Mediated Control of LIN28/let-7 Axis Contributes to Metabolic Adaptation of Breast Cancer Cells to Hypoxia.

Solid tumors, including breast cancer, are characterized by the hypoxic microenvironment, extracellular acidosis, and chemoresistance. Hypoxia marker, carbonic anhydrase IX (CAIX), is a pH regulator providing a selective survival advantage to cancer cells through intracellular neutralization while facilitating tumor invasion by extracellular acidification. The expression of CAIX in breast cancer patients is associated with poor prognosis and metastases. Importantly, CAIX-positive hypoxic tumor regions are enriched in cancer stem cells (CSCs). Here we investigated the biological effects of CA9-silencing in breast cancer cell lines. We found that CAIX-downregulation in hypoxia led to increased levels of let-7 (lethal-7) family members. Simultaneously with the increase of let-7 miRNAs in CAIX-suppressed cells, LIN28 protein levels decreased, along with downstream metabolic pathways: pyruvate dehydrogenase kinase 1 (PDK1) and phosphorylation of its substrate, pyruvate dehydrogenase (PDH) at Ser-232, causing attenuation of glycolysis. In addition to perturbed glycolysis, CAIX-knockouts, in correlation with decreased LIN28 (as CSC reprogramming factor), also exhibit reduction of the further CSC-associated markers NANOG (Homeobox protein NANOG) and ALDH1 (Aldehyde dehydrogenase isoform 1). Oppositely, overexpression of CAIX leads to the enhancement of LIN28, ALDH1, and NANOG. In conclusion, CAIX-driven regulation of the LIN28/let-7 axis augments glycolytic metabolism and enhances stem cell markers expression during CAIX-mediated adaptation to hypoxia and acidosis in carcinogenesis.

Cancer-associated S100P protein binds and inactivates p53, permits therapy-induced senescence and supports chemoresistance.

S100P belongs to the S100 family of calcium-binding proteins regulating diverse cellular processes. Certain S100 family members (S100A4 and S100B) are associated with cancer and used as biomarkers of metastatic phenotype. Also S100P is abnormally expressed in tumors and implicated in migration-invasion, survival, and response to therapy. Here we show that S100P binds the tumor suppressor protein p53 as well as its negative regulator HDM2, and that this interaction perturbs the p53-HDM2 binding and increases the p53 level. Paradoxically, the S100P-induced p53 is unable to activate its transcriptional targets hdm2, p21WAF, and bax following the DNA damage. This appears to be related to reduced phosphorylation of serine residues in both N-terminal and C-terminal regions of the p53 molecule. Furthermore, the S100P expression results in lower levels of pro-apoptotic proteins, in reduced cell death response to cytotoxic treatments, followed by stimulation of therapy-induced senescence and increased clonogenic survival. Conversely, the S100P silencing suppresses the ability of cancer cells to survive the DNA damage and form colonies. Thus, we propose that the oncogenic role of S100P involves binding and inactivation of p53, which leads to aberrant DNA damage responses linked with senescence and escape to proliferation. Thereby, the S100P protein may contribute to the outgrowth of aggressive tumor cells resistant to cytotoxic therapy and promote cancer progression.

Glucocorticoid receptor-mediated transcriptional activation of S100P gene coding for cancer-related calcium-binding protein.

S100P is a member of the S100 family of calcium-binding proteins involved in calcium sensing and signal transduction. Its abnormal expression and biological activities are linked to tumor phenotype, namely to increased survival, proliferation, invasion and metastatic propensity of tumor cells. Association of S100P with outcome of tumor treatment and preliminary data from S100P promoter analysis prompted us to study regulation of S100P expression by glucocorticoids, which are implicated in tumor response to chemotherapy. We showed that dexamethasone (DX), a representative glucocorticoid, was capable to induce activity of S100P promoter by means of increased expression, nuclear translocation, and transactivation properties of the glucocorticoid receptor (GR). Moreover, DX treatment led to decreased phosphorylation of ERK1/2, reduced transcriptional activity of AP1, and modulated activity of some additional transcription factors. We identified a promoter region responsible for DX-mediated transactivation and proved GR binding to S100P promoter. We found that the effect of DX was enhanced by partial but not complete inhibition of the MAPK/ERK pathway, supporting an active crosstalk between GR and MAPK/ERK signal transduction in control of S100P expression. On the other hand, suppression of GR mRNA level by transient siRNA expression resulted in reduced S100P transcription. The role of GR activation in S100P regulation was supported by co-expression of GR with S100P in cells treated with DX. These data suggest that S100P is a direct transcriptional target of glucocorticoid-mediated signaling in tumor cells that is activated through the interplay of GR and MAPK pathways.

Transcriptional regulation and functional implication of S100P in cancer.

S100P is an EF-hand calcium-binding protein that was originally identified in placenta and subsequently associated with cancer. It is a member of S100 family of proteins that function as extracellular and/or intracellular regulators of diverse cellular processes and participate in various human pathologies. S100P expression was detected in a spectrum of human tumor cell lines and tissues derived from breast, prostate, pancreas, lung and colon, where it was connected with malignant phenotype, hormone independence and resistance to chemotherapy. Overexpression of S100P was shown to promote tumorigenesis and metastasis in diverse cancer models. Functional studies of S100P indicate that its biological activities are exerted through extracellular signaling via RAGE receptor, resulting in increased proliferation and survival, or through intracellular interaction with ezrin, leading to increased cell migration and metastasis. Molecular mechanisms regulating expression of S100P in cancer cells are just emerging. Besides earlier described DNA methylation, recent studies implicate bone morphogenic protein and non-steroidal anti-inflammatory drugs in control of S100P expression during tumor progression. Functional analysis of S100P promoter identified SMAD, STAT/CREB and SP/KLF binding sites as key regulatory elements participating in transcriptional activation of S100P gene in cancer cells. Moreover, the most recent data reveal that expression of S100P is up-regulated by activation of glucocorticoid receptor suggesting that S100P could play a role in therapy resistance mediated by glucocorticoids in solid tumors. Elucidation of S100P regulation is an important step towards understanding biological significance of its tissue distribution and proposing strategies for targeted S100P modulation.

The induction of S100p expression by the Prostaglandin E2 (PGE2)/EP4 receptor signaling pathway in colon cancer cells.

BACKGROUND: Prostaglandin E2 (PGE2) levels are frequently elevated in colorectal carcinomas. PGE2 is perceived via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is most relevant. PGE2/EP4-receptor interaction activates CREB via the ERK/MEK pathway. However, the downstream target genes activated by this pathway remained to be investigated. METHODOLOGY/PRINICIPAL FINDINGS: Here, we have identified S100P (an EF-hand calcium binding protein) as a novel downstream target. We show by realtime RT-PCR that S100P mRNA levels are elevated in 14/17 (82%) colon tumor tissues as compared to paired adjacent normal colonic tissues. S100P expression is stimulated in the presence of PGE2 in a time dependent manner at mRNA and protein levels in colon, breast and pancreatic cancer cells. Pharmacological and RNAi-mediated inhibition of the EP4 receptor attenuates PGE2-dependent S100P mRNA induction. RNA(i)-mediated knockdown of CREB inhibits endogenous S100P expression. Furthermore, using luciferase reporter analysis and EMSA we show that mutation and/or deletion of the CRE sequence within the S100P promoter abolished PGE2-mediated transcriptional induction. Finally, we demonstrate that RNA(i)-mediated knockdown of S100P compromised invadopodia formation, colony growth and motility of colon cancer cells. Interestingly, endogenous knock down of S100P decreases ERK expression levels, suggesting a role for ERK in regulating S100P mediated cell growth and motility. CONCLUSIONS/SIGNIFICANCE: Together, our findings show for the first time that S100P expression is regulated by PGE2/EP4-receptor signaling and may participate in a feedback signaling that perpetuates tumor cell growth and migration. Therefore, our data suggest that dysregulated S100P expression resulting from aberrant PGE2/EP4 receptor signaling may have important consequences relevant to colon cancer pathogenesis.

Key promoter elements involved in transcriptional activation of the cancer-related gene coding for S100P calcium-binding protein.

S100P gene encodes a calcium-binding protein expressed in different tumor tissues and is functionally implicated in malignant phenotype. Despite consistent relationship to cancer, regulation of S100P gene expression has remained unexplored. Here we determined the transcription start and defined the S100P core promoter. Using a series of the promoter constructs analyzed by dual luciferase reporter assay, we identified SMAD, STAT/CREB and SP/KLF binding sites as critical cis-elements required for S100P expression in cancer cells. We also demonstrated in EMSA that these elements bind nuclear factors, and showed their functional significance by promoter deletion analysis. This study represents the first coherent contribution to understanding of factors and pathways responsible for S100P gene activation in cancer.

The calcium-binding protein S100P in normal and malignant human tissues.

BACKGROUND: S100P is a Ca2+ binding protein overexpressed in a variety of cancers, and thus, has been considered a potential tumor biomarker. Very little has been studied about its normal expression and functions. METHODS: We examined S100P expression in normal human tissues by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. S100P protein expression was also studied in a series of tumors, consisting of 74 ovarian, 11 pancreatic, 56 gastric, 57 colorectal, 89 breast and 193 prostate carcinomas using a novel anti-S100P monoclonal antibody. RESULTS: Among the normal tissues, the highest S100P mRNA levels were observed in the placenta and esophagus. Moderate signals were also detected in the stomach, duodenum, large intestine, prostate and leukocytes. At the protein level, the highest reactions for S100P were seen in the placenta and stomach. Immunostaining of tumor specimens showed that S100P protein is expressed in all the tumor categories included in the study, being most prevalent in gastric tumors. CONCLUSION: Based on our observations, S100P is widely expressed in both normal and malignant tissues. The high expression in some tumors suggests that it may represent a potential target molecule for future diagnostic and therapeutic applications.

Expression of S100P protein correlates with and contributes to the tumorigenic capacity of HeLa cervical carcinoma cells.

Tumor growth is associated with multiple changes at the gene expression level. Recognition of the genes differentially expressed between the cellular populations at various degrees of malignancy may provide valuable clues towards the identification of clinically useful diagnostic markers and/or therapeutic targets. In the present study, we used suppression subtractive PCR to identify differentially expressed genes with possible relevance for control of tumorigenic potential using two cervical carcinoma cell lines of the common HeLa origin, but of different capacity to generate tumors in nude mice. Screening of the subtracted libraries resulted in isolation of several known as well as novel genes including the gene encoding S100P calcium-binding protein that belongs to S100 family, whose members can bind and modulate effector proteins in a calcium-dependent manner. Expression of S100P was further studied in the context of different culture conditions and was found to correlate with the tumorigenic phenotype of the somatic cell hybrids between HeLa and normal human fibroblasts. Moreover, S100P was highly expressed in a number of tumorigenic cell lines derived from colorectal and breast carcinoma, suggesting that it is not restricted to a particular tumor type. Functional involvement of S100P in tumor growth was evaluated using tumor xenografts produced from the cells transfected with the full-length S100P cDNA. The results showed that S100P can positively affect anchorage-independent growth of the transfected cells and improve tumor formation in nude mice, suggesting that it actively participates in the control of the tumorigenic potential in vivo.

Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH.

Acidic extracellular pH (pHe) is a typical attribute of a tumor microenvironment, which has an impact on cancer development and treatment outcome. It was believed to result from an accumulation of lactic acid excessively produced by glycolysis. However, metabolic profiles of glycolysis-impaired tumors have revealed that CO2 is a significant source of acidity, thereby indicating a contribution of carbonic anhydrase (CA). The tumor-associated CA IX isoform is the best candidate, because its extracellular enzyme domain is highly active, expression is induced by hypoxia and correlates with poor prognosis. This study provides the first evidence for the role of CA IX in the control of pHe. We show that CA IX can acidify the pH of the culture medium in hypoxia but not in normoxia. This acidification can be perturbed by deletion of the enzyme active site and inhibited by CA IX-selective sulfonamides, which bind only to hypoxic cells containing CA IX. Our findings suggest that hypoxia regulates both expression and activity of CA IX in order to enhance the extracellular acidification, which may have important implications for tumor progression.

Monoclonal antibodies generated in carbonic anhydrase IX-deficient mice recognize different domains of tumour-associated hypoxia-induced carbonic anhydrase IX.

Transmembrane carbonic anhydrase IX (CA IX) is frequently expressed in human tumours in response to hypoxia and may serve as a tumour marker and therapeutic target. So far, only a single monoclonal antibody (MAb) M75 with an epitope in the N-terminal proteoglycan (PG)-like region has been available for detection purposes. Attempts to produce MAbs against other parts of CA IX were unsuccessful due to the immunodominance of the PG region that significantly differs between human and mouse homologues. To overcome this problem, we used various forms of human CA IX antigen to immunize CA IX-deficient mice recently produced by targeted disruption of Car9 gene. Here, we describe new MAbs that react with human, but not mouse CA IX in different immunodetection settings, and show no cross-reactivity with CA I, II and XII. MAb IV/18 is directed to the PG region, while the other six antibodies bind to the CA domain, as determined by CA IX deletion variants. IV/18 recognizes a linear epitope, while anti-CA MAbs V/10, V/12, VII/20, VII/28, VII/32 and VII/38 react with conformational epitopes clustered into three antigenic sites. The new antibodies represent important tools for improving our knowledge of structure-function relationships in the CA IX molecule and a better understanding of the role of CA IX in cancer development. Moreover, the availability of the MAbs specific for distinct antigenic regions on two separate extracellular domains offers an opportunity to elaborate a sensitive assay that could be particularly important for CA IX detection in body fluids of cancer patients.

Carbonic anhydrase IX reduces E-cadherin-mediated adhesion of MDCK cells via interaction with beta-catenin.

Carbonic anhydrase IX (CA IX) is a cancer-associated transmembrane isoform of zinc metalloenzymes that catalyse interconversion between carbon dioxide and bicarbonate. CA IX is strongly induced by tumor hypoxia and has been proposed to participate in acidification of tumor microenvironment and in cell adhesion. To elucidate the cell adhesion-related role of CA IX, we investigated its subcellular localization and relationship to E-cadherin, a key adhesion molecule whose loss or destabilization is linked to tumor invasion. For this purpose, we generated MDCK cells with constitutive expression of human CA IX protein. During the monolayer formation, CA IX was localized to cell-cell contacts and its distribution in lateral membranes overlapped with E-cadherin. Calcium switch-triggered disruption and reconstitution of cell contacts resulted in relocalization of both CA IX and E-cadherin to cytoplasm and back to plasma membrane. A similar phenomenon was observed in hypoxia-treated and reoxygenated cells. Moreover, CA IX-expressing MDCK cells exhibited reduced cell adhesion capacity and lower levels of Triton-insoluble E-cadherin. Finally, CA IX was found to coprecipitate with beta-catenin. We conclude that CA IX has a capacity to modulate E-cadherin-mediated cell adhesion via interaction with beta-catenin, which could be of potential significance in hypoxia-induced tumor progression.

Gastric hyperplasia in mice with targeted disruption of the carbonic anhydrase gene Car9.

BACKGROUND & AIMS: Carbonic anhydrase (CA) IX is a highly active enzyme with adhesion capacity that is functionally implicated in acid-base balance and intercellular communication. It is normally present in basolateral membranes of gastrointestinal epithelial cells and ectopically expressed in various carcinomas. To show its physiologic relevance, we have cloned the Car9 gene and generated CA IX-deficient mice. METHODS: The mice with null mutation of the Car9 gene were obtained by targeted gene disruption. Tissue architecture and expression of markers were determined by histochemical and immunohistochemical techniques. RESULTS: Mice homozygous for the mutation developed gastric hyperplasia of the glandular epithelium with numerous cysts. The first changes were observed in the newborn animals, and the hyperplasia became prominent at the end of gastric morphogenesis in 4-week-old mice. Loss of CA IX led to overproduction of mucus-secreting pit cells and depletion of pepsinogen-positive chief cells. The proportion of H(+)/K(+)-adenosine triphosphatase-positive parietal cells significantly decreased, but their absolute number was not reduced. Correspondingly, CA IX-deficient mice had normal gastric pH, acid secretion, and serum gastrin levels. CONCLUSIONS: Phenotypic consequences of the Car9 null mutation show the important role of CA IX in morphogenesis and homeostasis of the glandular gastric epithelium via the control of cell proliferation and differentiation.