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Carcinogenesis Advance Access originally published online on June 4, 2007
Carcinogenesis 2007 28(10):2096-2104; doi:10.1093/carcin/bgm124
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

MAP17 enhances the malignant behavior of tumor cells through ROS increase

Maria V. Guijarro, Juan F.M. Leal, Carmen Blanco-Aparicio, Soledad Alonso1, Jesús Fominaya, Matilde Lleonart2, Josep Castellvi2, Santiago Ramon y Cajal2 and Amancio Carnero*

Experimental Therapeutics Program
1 Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas, Melchor Fernandez Almagro, 3, 28029 Madrid, Spain
2 Departmento Anatomía Patológica, Hospital Vall d'Hebrón 08035, Barcelona, Spain

* To whom correspondence should be addressed. Tel: +34 91 732 8021; Fax: +34 91 224 6976; Email: acarnero{at}cnio.es


   Abstract
Top
 Abstract
Introduction
 Experimental procedures
 Results
 Discussion
 Funding
 References
 
Tumorigenesis occurs when the mechanisms involved in the control of tissue homeostasis are disrupted and cells stop responding to physiological signals. Therefore, genes capable of desensitizing tumoral cells from physiological signals may provide a selective advantage within the tumoral mass and influence the outcome of the disease. We undertook a large-scale genetic screen to identify genes able to alter the cellular response to physiological signals and provide selective advantage once tumorigenesis has begun. We identified MAP17, a small 17 kDa non-glycosylated membrane protein previously identified by differential display being over-expressed in carcinomas. Tumor cells that over-express MAP17 show an increased tumoral phenotype with enhanced proliferative capabilities both in presence or absence of contact inhibition, decreased apoptotic sensitivity and increased migration. MAP17-expressing clones also grow better in nude mice. The increased malignant cell behavior induced by MAP17 are associated with an increase in reactive oxygen species (ROS) production, and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells. Treatment of melanoma cells with inhibitors of Na+-coupled co-transporters lead to an inhibition of ROS increase and a decrease in the malignant cell behavior in MAP17-expressing clones. Finally, we show that MAP17-dependent ROS increase and tumorigenesis are dependent on its PDZ-binding domain, since disruption of its sequence by point mutations abolishes its ability to enhance ROS production and tumorigenesis. Our work shows the tumorigenic capability of MAP17 through a connection between Na+-coupled co-transporters and ROS.

Abbreviations: FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; ROS, reactive oxygen species; MAP17, membrane associated protein 17 kDa


   Introduction
Top
 Abstract
 Introduction
Experimental procedures
 Results
 Discussion
 Funding
 References
 
Although multiple oncogenes that activate signaling pathways directly involved in cell survival or proliferation have been discovered in the last decades, many other genes that provide an advantage to the tumoral cells making them insensitive to physiological signals or altering their normal physiology are still to be found. Functional genetic screens using retroviral delivery of high complexity cDNA libraries constitute a valuable tool to discover new genes involved in the appropriate phenotypic characteristic of the tumorigenic process (1–3). A genome-wide retroviral cDNA screen to search for genes that confer cancer cell selective advantage during tumorigenesis allowed us to identify MAP17 (4), a small non-glycosylated membrane-associated protein of 17 kDa that locates to the plasma membrane and the Golgi apparatus (5,6). The protein sequence showed a hydrophobic N-terminus of 13 amino acids encoding a PDZ-binding domain and two transmembrane regions (7,8). MAP17 was first described by differentital display over-expressed in carcinomas (5). Transfection of full-length wild-type MAP17 into HT29 colon carcinoma cells decreased cell proliferation in vitro and tumor growth in vivo (6). MAP17 binds several PDZ domain-containing proteins, including PDZK1, NHERF proteins, NaPiIIa and NHe3. Over-expression of MAP17 into opossum kidney cells participates together with NHRF3 and NHRF4 in NaPiIIa internalization to the transgolgi network (8). In a transgenic mouse model, MAP17 hepatic over-expression resulted in PDZK1 liver deficiency, suggesting that MAP17 is an endogenous regulator of PDZK1 turnover (9). MAP17 acts as an atypical anchoring site for PDZK1 and interacts with the NaPi-Iia–PDZK1 protein complex in renal proximal tubular cells (10). The physiological role of MAP17 in proximal tubules is not known but it stimulates specific Na-dependent transport of mannose and glucose in Xenopus oocytes (11). MAP17 shares regulatory elements with the stem cell leukemic gene (TAL-1) a basic HLH protein essential in the formation of the hematopoietic lineages (12,13), although major expression of MAP17 has been found only in kidney among normal tissues.

MAP17 is over-expressed, mostly through mRNA amplification, in a great variety of human carcinomas (5,14). Immunohistochemical analysis of MAP17 during cancer progression shows, at least in prostatic and ovarian carcinomas, that over-expression of the protein strongly correlates with tumoral progression. Many tumor cells also express MAP17 and its expression does not correlate with expression of stem cell leukemic gene, a neighbor gene reported to be co-expressed in some hematopoietic cell lines. Stem cell leukemic gene neither is expressed in most MAP17-positive tumors, indicating the independent transcription of MAP17, al least in carcinomas (14). Moreover, we have found that MAP17 promoter is activated by oncogenes (14).

However, the known facts' data do not provide an explanation why MAP17 could be over-expressed in some carcinoma tumors or the reason to be selected in a phenotypic screen providing selective advantage during tumorigenesis. In the present work, we describe how MAP17 enhances the tumorigenic properties of tumoral cells, providing them a selective advantage during cancer progression. Moreover, our work shows a connection between membrane pumps and reactive oxygen species (ROS) and their role increasing tumorigenic properties of melanoma cells.


   Experimental procedures
Top
 Abstract
 Introduction
 Experimental procedures
Results
 Discussion
 Funding
 References
 
Cell culture
A375 malignant melanoma cells from American Type Culture Collection were maintained in Dulbecco's modified Eagle's medium (DMEM) with glutamax (Gibco) containing 10% fetal bovine serum (FBS) (Sigma), penicillin, streptomycin and fungizone. Cultures were selected when indicated with 75 µg/ml hygromycin (Calbiochem), 400 µg/ml G418 (Sigma) or 2 µg/ml puromycin (Fluka). To perform the gene transfer using ecotropic competent retroviruses, cells were transfected with the murine receptor and a mass culture expressing the receptor used for the following experiments. Human MAP17 full cDNA and mutant were cloned in pBabepuro and clones generated by gene transfer. Numerous clones were selected and tested. All behave similarly. For this work, four representative clones were used.

Retroviral-mediated gene transfer
Packaging LinXE cells were plated in a 10 cm dish, incubated for 24 h and then transfected by calcium phosphate precipitation with 20 µg of the retroviral plasmid (16 h at 37°C). After 48 h, the virus-containing medium was filtered (0.45 µm filter, Millipore) and supplemented with 8 µg/ml polybrene (Sigma) and an equal volume of fresh medium. Target cells were seeded at 106 cells per 10 cm dish and incubated overnight. For infections, the culture medium was replaced by the appropriate viral supernatant; the culture plates were centrifuged (1 h, 1500 r.p.m.) and incubated at 37°C for 16 h. The infected cell population was purified using the appropriate selection.

Reverse transcription–polymerase chain reaction
Total RNA was purified using the TRI-REAGENT (Molecular Research Center, Cincinnati, OH). Reverse transcription was performed with 5 µg of mRNA using MMLV reverse transcriptase (Promega) and oligodT primer according to the manufacturer's recommendations. The following primers were used to amplify specific cDNA regions: MAP17, forward 5'-CAGCCATGTCGGCCCTCA-3' and reverse 5'-TTATTTCACAGAAATTAGGGCC-3' and ß-actin, forward 5'-AGGCCAACCGCGAGAAGATGAC-3' and reverse 5'-GAAGTCCAGGGCGACGTAGCA-3'. cDNA was subjected to polymerase chain reaction under standard conditions (95°C 30 s, 65°C 45 s and 72°C 45 s; 30 cycles) and products were analyzed by 1% agarose gel electrophoresis.

Surrogated assays
Growth in low serum.
A time course curve of parental and MAP17-expressing cells was generated by seeding 104 cells in 2.5 cm dishes in triplicate samples. After 24 h, medium was changed (day 0) and the indicated culture media added. After 4 days, cells were fixed and stained with crystal violet. After extensive washing, crystal violet was resolubilized in 15% acetic acid and quantified at 595 nm as a relative measure of cell number. Values are expressed as the percentage of cell growth of cells growing in the presence of 10% FBS. Zero percent refers to the number of cells at day 0. Culture media tested were as follows: NS (no serum or complements used, cells were grown in DMEM alone), 0.5% (DMEM + 0.5% FBS), 0.5% + ITS (DMEM + 0.5% FBS + insulin + transferrin and selenium), ITS (DMEM + insulin + transferrin and selenium).

Doubling time.
A time course curve of parental and MAP17-expressing cells growing in the presence of serum was generated. Doubling time was calculated from the exponential growth of the different cultures.

Clonability.
Cells (102, 5 x 102, 103 and 104) were seeded in 10 cm plates. The medium was replaced every 3 days and after 10 days the cells were fixed and stained with crystal violet. After extensive washing, colonies were counted. Values are expressed as the percentage of colonies related to the parental cell line.

Growth in soft agar.
To measure the anchorage-independent growth, 2 x 104 cells were suspended in 1.4% agarose D-1 Low EEO (Pronadisa) growth medium containing 10% FBS, disposed onto a solidified base of growth medium containing 2.8% agar (agarose D-1 Low EEO, Pronadisa) and overlaid with 1 ml of growth medium. After 24 h, media containing 10% FBS were added to each 35 mm dish and renewed twice weekly. Colonies were scored 3 weeks after and all values were determined in triplicate. Photographs were taken with a phase-contrast microscope (Olympus).

Wound-healing assays.
An artificial ‘wound’ was created using a 10 µl pipette tip on confluent cell monolayer in four-chamber plates (Lab-Tek II chambered 1.5 cover glass system 155382, Nalge Nunc International) in complete medium. Photographs were taken every 10 min during 12 h using a confocal ultra-spectral microscope Leica TCS-SP2-AOBS-UV.

MAP17 protein determination
Cells (106) were re-suspended in 100 µl of phosphate-buffered saline (PBS). Cells were fixed and permeabilized to give the antibody access to intracellular structures according to the manufacturer's recommendations (Fix and Perm; Caltag GAS001). Cells were then labeled with monoclonal anti-MAP17 antibody for 20 min at room temperature, washed with PBS and incubated with R-phycoerythrin-conjugated goat anti-mouse IgG (Molecular Probes). After washing with PBS, cells were re-suspended in the appropriate buffer and analyzed with the FACScalibur Becton and Dickinson Flow Cytometer.

Monoclonal MAP17 antibody was generated from bacterial purified GST-MAP17 protein (14). Several clonal antibodies were tested for specificity and validated by antigen competition. Similar results were obtained with at least other three anti-MAP17 monoclonal antibodies.

ROS fluorescent detection
To visualize intracellular ROS levels, A375 cells grown on cover slips were washed twice with warm PBS and then incubated at 37°C with 8 µM of CM-H2DCFDA (5-(6)chloromethyl-2'7' dichloro dihydrofluorescein diacetate acetyl ester) in warm PBS supplemented with 2.5 mM glucose for 15 min. Then, PBS was replaced with DMEM supplemented wit 10% FBS and cells were incubated 10 min further in the same conditions. Cells were washed once again with warm PBS and fixed with 4% paraformaldehyde (Sigma) at room temperature for 5 min. The fixed cells were washed three times with PBS and cover slips mounted in mowiol. Intracellular ROS were visualized using a confocal ultra-spectral microscope Leica TCS-SP2-AOBS-UV.

Xenograft in nude mice
Tumorigenicity was assayed by the subcutaneous injection of 4 x 106 cells into the back legs of 4-week-old female athymic nude mice. Animals were examined weekly and incubated for 8 weeks. Tumors were measured using calipers. Tumor volume (mm3) was determined using the standard formula a2*b/2, where a is the width and b is the length. All animal experiments were done under the experimental protocol approved by Spanish National Cancer Center's (CNIO's) Institutional Committee for Care and Use of Animals.


   Results
Top
 Abstract
 Introduction
 Experimental procedures
 Results
Discussion
 Funding
 References
 
MAP17 confers proliferative advantage in full serum conditions to human melanoma cells
To study the biological effect of MAP17, we selected human tumor cells that do not express MAP17 mRNA, expressed the wild-type human MAP17 cDNA and studied the alteration of the biological properties of these cells. To that end, we selected A375 from human melanoma cells. First, we generated clones from A375 human melanoma cells ectopically expressing the full human MAP17 cDNA driven by a LTR promoter. We selected several clones and characterized the MAP17 mRNA expression by reverse transcription–polymerase chain reaction (Figure 1A). All the clones analyzed expressed considerable amounts of MAP17 mRNA in contrast to the parental A375 cells. To analyze the protein level, we generated mouse monoclonal antibodies using as immunogen, the purified recombinant MAP17 produced in Escherichia coli. We quantitated MAP17 protein in cells by FACscan. Growing cells from the parental A375 line, expressing the empty vector, and from each MAP17-expressing clone were permeabilized and incubated with the MAP17 antibody conjugated with a fluorophore. FACs analysis showed that all the clones expressed much higher level of MAP17 than parental cells (Figure 1B). We confirmed these data by immunohistochemistry and immunofluorescence (Figure 1C).


Figure 1
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  		Fig. 1. Characterization of MAP17-expressing clones. (A) Characterization of MAP17-expressing clones by reverse transcription–polymerase chain reaction. A375 melanoma cells were transfected with exogenous MAP17 cDNA in a pBabepuro vector. Individual clones were selected and analyzed for MAP17 mRNA expression by reverse transcription–polymerase chain reaction as indicated in Experimental procedures. Four representative clones (C1, C2, C3 and C4) and cells carrying empty vector (P) are shown. (B) MAP17 protein level determination. To analyze cellular levels of MAP17 protein, cells were incubated with an anti-MAP17 primary antibody and R-phycoerythrin-conjugated goat anti-mouse secondary antibody and 104 cells from each clone and parental cells were analyzed with a FACScalibur flow cytometer. MAP17 content is represented by MAP17-associated fluorescence (x-axis) and each cell (y-axis). Four representative clones (C1, C2, C3 and C4) and cells carrying empty vector (P) are shown. (C) Characterization of MAP17-expressing clones by immunohistochemistry and immunofluorescence. A375 melanoma cells were transfected with exogenous MAP17 cDNA in pBabepuro vector. Individual clones were selected and analyzed for MAP17 protein expression by immunodetection by immunohistochemistry (upper figures) and immunofluorescence (bottom figures). Three representative clones (C2, C3 and C4) and parental (P) cells are shown.

 
Next, we checked whether MAP17 increased the oncogenic potential of A375 cells. To that end, we measured several tumorigenic properties in A375 parental cells and MAP17-expressing clones. First, we compared their doubling times during exponential growth. MAP17-expressing clones grew faster than parental cells in 10% fetal calf serum-supplemented medium decreasing their doubling time (Figure 2A). Analysis of the cell cycle distribution by flow cytometry showed a moderate increase in the percentage of cells in S phase induced by ectopic expression of MAP17 (Figure 2B). However, MAP17-expressing clones did not show altered growth in medium supplemented with 0.5% fetal calf serum, ITS (insulin, transferrin and selenium) or without serum (data not shown). This data indicate that MAP17 confers proliferative advantage when cells are growing in full serum conditions.


Figure 2
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  		Fig. 2. MAP17 expression enhances the tumorigenic properties of A375 tumor cells. (A) MAP17 expression decreases doubling time. A growth curve of both parental cells (cells carrying empty vector) and MAP17-expressing clones was performed following the procedure described in Experimental procedures. Doubling time for each population was calculated while the cells where in exponential growth. Four representative clones (C1, C2, C3 and C4) and parental cells (cells carrying empty vector, P) are shown. Data presented are the mean from three independent experiments. (B) MAP17 expression increases the percentage of cells in S phase. Asynchronously growing cells (104) from each clone and parental cells were analyzed for DNA content with a FACScalibur flow cytometer. Data show results from parental (cells carrying empty vector) and three representative clones (C2, C3 and C4) of one representative experiment out of three performed. (C) MAP17 increases survival of cells seeded at very low density. Cells (103) were seeded in 10 cm dishes in triplicate and grown for 10 days, then fixed and stained with crystal violet and number of colonies counted. Four representative clones (C1, C2, C3 and C4) and parental cells (cells carrying empty vector, P) are shown. Data presented are the mean from triplicate samples; bars, ±SD. (D and E) MAP17 expression increases colony number and size in soft agar. Cells (104) were cultured following the procedure described in Experimental procedures, 4 weeks later they were fixed and pictures taken. Four representative clones (C1, C2, C3 and C4) and parental cells (P) are shown. (E) Quantification, number of colonies (bold). Data presented are the mean from triplicate samples; bars, ±SD. Colony size (hatched) was determined by measuring a minimum of 30 representative colonies. (F and G) MAP17 expression enhances migration. Cells were cultured in four-chamber plates until 100% confluence was reached. Wound was performed and a picture was taken every 4 h. Four representative clones (C1, C2, C3 and C4) and parental cells (P) were performed with similar results. Only clone C4 and parental cells are shown. (G) Quantification. The wound area filled was calculated from several samples (from F) and presented as a mean; bars, ±SD.

 
To determine their ability to overcome apoptosis in the absence of cell contact, we measured the capability to form colonies when cells were seeded at very low density. We plated 103 cells per 10 cm dish and cultured them during 1 week in complete medium. MAP17-expressing cells formed 2- to 4-fold more colonies in these conditions than parental A375 cells expressing only the vector (Figure 2C), indicating that MAP17 enhanced the survival of these cells in the absence of cell contact.

We also determined the ability of the clones to grow in anchorage-independent conditions. We seeded 104 cells from parental or MAP17-expressing clones in soft agar and cultured them for 3 weeks. Whereas parental A375 cells produced only few small colonies, MAP17-expressing clones produced a high number of larger colonies (Figure 2D and E).

To check whether MAP17 modifies the migration capability of the cells, we performed an in vitro wound-filling assay. Cells were seeded in four-chamber slides and once they reached confluence we generated a wound in the cell monolayer and measured their ability to close the wound. MAP17-expressing clones showed a greatly increased ability to migrate and to close the wound (Figure 2F and G).

To confirm these properties in a more physiological setting, we injected parental A375 cells or MAP17-expressing clones into nude mice and followed the growth of the xenografts. MAP17-expressing tumors grew faster, reaching a larger size than parental A375 over the same period of time (Figure 3), confirming the potential oncogenicity of MAP17 protein. The immunohistochemistry analysis of the tumoral mass confirmed MAP17 expression in the tumors arising from the clones (Figure 3C and data not shown).


Figure 3
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  		Fig. 3. MAP17 expression enhances xenograft growth. Parental (P) or MAP17-expressing cells (Clones C1, C2, C3 and C4) were injected in nude mice in duplicate and grown during 8 weeks. Tumors were measured once a week during 8 weeks and then extracted. (A) Data presented are the average volume of the tumors from two independent experiments at 8 weeks after injection. (B) Represertative picture of tumor size of Parental or one MAP17 expressing clone (24). Tumors were processed for immunostaining (C) with anti-MAP17 antibodies. Similar data were obtained with other MAP17-espressing clones. Statistics were calculated using the Mann–Whitney test for n = 4. In all cases P = 0.286.

 
Thus, the ectopic expression of MAP17 in melanoma cells is associated with the enhancement of malignant cell behavior, indicating that MAP17 is implicated in growth rate, survival in the absence of cell contact, anchorage-independent growth, motility and tumorigenicity in nude mice.

The enhanced oncogenic capabilities induced by MAP17 in tumoral cells are due to an increase of intracellular ROS
Growing evidence suggests that ROS act as second messengers in intracellular signaling cascades that induce and maintain the oncogenic phenotype of tumoral cells. ROS have been described to induce proliferation, survival and cellular migration (15–17). We have observed that MAP17 is able to induce similar properties in tumor cells; therefore, we investigated whether MAP17 expression induces an increase of intracellular ROS. First, we measured the level of ROS in living cells with the fluorescent probe DCF (2,7, Dichlorofluorescein). MAP17-expressing clones showed a clear increase of ROS respect to their parental counterparts (Figure 4A). To confirm this point in the whole population, we measured superoxide generation by DCF in the conditions indicated in Experimental procedures. Then, we analyzed by FACscan the level of DCF fluorescence in a total of 104 cells. MAP17-expressing clones have, in average, 30% more intracellular ROS than parental cells. This increase in ROS levels was inhibited by glutathione (Figure 4B), the most important low molecular size thiol involved in cellular detoxification, redox balance and stress response, and by N-acetyl-cysteine, another important ROS scavenger (Figure 4B).


Figure 4
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  		Fig. 4. MAP17-enhanced tumorigenicity depends on an intracellular increase in ROS. (A) MAP17 increases ROS. To visualize intracellular ROS levels, A375 parental or MAP17-expressing cells were grown on cover slips, and then incubated at 37°C with 8 µM of CM-H2DCFDA as described in Experimental procedures. Intracellular ROS were visualized using a confocal ultra-spectral microscope Leica TCS-SP2-AOBS-UV. A representative picture of MAP17-expressing clones and parental cells is shown. (B) ROS levels are reduced by glutathione and N-acetyl-cysteine. The above populations were analyzed by FACs and the relative levels of CM-H2DCFDA fluorescence represented in the individual clones and parental cells. Ten thousand cells were counted in each sample. To study the levels of ROS in the presence of scavengers, cells were treated with 10 mM glutathione or 10 mM N-acetyl-cysteine for 4 h previous the analysis. (Cand D) MAP17 enhances cell survival through ROS production. (C) Colony number: triplicate cell samples were cultured for 10 days in the presence or absence of 10 mM glutathione or N-acetyl-cysteine, and then fixed and stained with crystal violet following the protocol described in Experimental procedures. Number of colonies counted. Four representative clones (C1, C2, C3 and C4) and parental cells (cells carrying empty vector, P) are shown. Data presented are the mean from triplicate samples; bars, ±SD. (D) Colony size: the diameter of 30 colonies was measured per sample and the average represented. Four representative clones (C1, C2, C3 and C4) and parental cells (P) are shown. Data presented are the mean from triplicate samples; bars, ±SD. (E and F) MAP17 increases colony number and size in soft agar through ROS production. Cells were grown in the presence or absence of 10 mM glutathione or N-acetyl-cysteine for 4 weeks then fixed, number of colonies counted. Presented data are the mean from three independent experiments; bars, ±SD. Four representative clones and parental cells (cells carrying empty vector, P) are shown. (F) Colony size was determined by measuring a minimum of 30 representative colonies. Presented data are the mean from three independent experiments; bars, ±SD. Four representative clones (C1, C2, C3 and C4) and parental cells (cells carrying empty vector, P) are shown. (G) MAP17 enhances migration through ROS. Cells were cultured until 100% confluence, and then medium changed to continue the experiment either in presence or absence of 10 mM glutathione. Wounds were performed and a picture taken every 4 h. The wound area filled was calculated from several samples and presented as a mean; bars, ±SD. Four representative clones (C1, C2, C3 and C4) and parental cells (cells carrying empty vector, P) were performed with similar results. Only clone C4 and parental cells are shown.

 
In transformed fibroblasts, Ras and Rac appear to be directly linked to the production of superoxide anions and in turn, transformation of these cells depends on ROS formation since it can be suppressed by treatment with antioxidants (18). We checked whether some of the transforming properties of MAP17 were linked to the detected intracellular ROS increase. To that end, we examined clonability, growth in soft agar and in vitro wound filling in the presence of antioxidants. We found that the presence of antioxidants reduced the oncogenic properties of the MAP17-expressing cells: they have reduced colony formation efficiency in the presence of antioxidants (Figure 4C) and the average colony size is dramatically reduced as an indication of reduced growth rate (Figure 4D). Interestingly, the colony formation efficiency and colony size are similar to parental cells in the absence of antioxidants. The same effect was found in the soft agar growth assay: antioxidants reduced the number and size of colonies of MAP17-expressing cells (Figure 4E and F). Finally, the presence of antioxidants inhibits the ability to migrate and fill the wound in MAP17-expressing cells (Figure 4G).

Our data indicate that the expression of MAP17 in human melanoma cells induces an increase of intracellular ROS that plays an important role in the enhancement of the malignant cell behavior.

MAP17-dependent ROS increase and tumorigenesis are dependent on its PDZ-binding domain
MAP17 structure is reduced to two transmembrane regions and one PDZ-binding domain. MAP17 binds several PDZ domain-containing proteins, including PDZK1, NHERF proteins, NaPiIIa and NHe3. To study whether the effect of MAP17 over-expression was related to its ability to bind PDZ domain-containing proteins, we generated a mutant MAP17 protein in which two amino acids out of the four comprising the PDZ-binding domain were mutated disrupting the structure (Figure 5A). We generated individual clones carrying mutant MAP17 (Figure 5B and C) and measured its ability to enhance the tumorigenic properties of A375 cells. MAP17 defective in its PDZ-binding domain was unable to increase growth rate, clonability or growth in soft agar (Figure 5D–G). These results confirm that the biological effect produced by MAP17 was dependent on its PDZ-binding domain.


Figure 5
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  		Fig. 5. MAP17-dependent ROS increase and tumorigenesis are dependent on its PDZ-binding domain. (A) Scheme of the changes made in the PDZ-binding domain of MAP17. (B) Characterization of MAP17 mutant-expressing clones by reverse transcription–polymerase chain reaction. A375 melanoma cells were transfected with exogenous MAP17 mutant cDNA in a pBabepuro vector. Individual clones were selected and analyzed for MAP17 mRNA expression by reverse transcription–polymerase chain reaction as indicated in Experimental procedures. Three representative clones (M1, M2 and M3) and parental (cells carrying empty vector, P) cells are shown. (C) Characterization of MAP17 mutant-expressing clones by immunohistochemistry. A375 melanoma cells were transfected with exogenous MAP17 cDNA in pBabepuro vector. Individual clones were selected and analyzed for MAP17 protein expression by immunodetection by immunohistochemistry. Three representative clones (M1, M2 and M3) and parental (cells carrying empty vector, P) cells are shown. (D) PDZ-binding mutant MAP17 do not enhances growth rate. A growth curve of both parental cells and MAP17-expressing clones was performed following the procedure described in Experimental procedures. Data presented are the mean from triplicate samples; bars, ±SD. Three representative clones (M1, M2 and M3) and parental cells (P) are shown. (E) PDZ-binding mutant MAP17 do not enhances cell survival through ROS production. Colony number: triplicate cell samples were cultured for 10 days in the presence or absence of 10 mM glutathione, and then fixed and stained with crystal violet following the protocol described in Experimental procedures. Number of colonies counted. Data presented are the mean from triplicate samples; bars, ±SD. Three representative clones (M1, M2 and M3) and parental cells (P) are shown. (D, F and G) PDZ-binding mutant MAP17 do not increases colony number and size in soft. Cells were grown in the presence or absence of 10 mM glutathione for 4 weeks then fixed, number of colonies counted (F). Presented data are the mean from three independent experiments; bars, ±SD. Three representative clones (M1, M2 and M3) and parental cells (P) are shown. (G) Colony size was determined by measuring a minimum of 30 representative colonies. Presented data are the mean from three independent experiments; bars, ±SD. Three representative clones (M1, M2 and M3) and parental cells (P) are shown. (H) PDZ-binding mutant MAP17 do not increases ROS. To visualize intracellular ROS levels, A375 parental or MAP17-expressing cells were grown on cover slips, and then incubated at 37°C with 8 µM of CM-H2DCFDA as described in Experimental procedures. Intracellular ROS were visualized using a confocal ultra-spectral microscope Leica TCS-SP2-AOBS-UV. Representative pictures of a MAP17-expressing clone (C4), two mutant clones (M1 and M3) and parental cells (cells carrying empty vector, P) are shown.

 
We have also shown that this effect is also dependent on ROS increase. Therefore, we measured whether both effects were linked. We measured ROS levels in A375-expressing mutated MAP17. MAP17 defective in its PDZ-binding domain was unable to increase intracellular ROS (Figure 5H).

Inhibition of Na+-coupled co-transporters inhibits MAP17-dependent ROS increase and tumorigenesis
MAP17 stimulates specific Na+-dependent transport of mannose and glucose in Xenopus oocytes (11) and this activity is sensitive to known inhibitors of Na+-coupled co-transporters (19). Furosemide acts by inhibiting the Na-K-2Cl symporter whereas phloridzin inhibits the Na+-coupled glucose transporters. Therefore, we tested whether MAP17-dependent ROS increase was dependent on Na+-coupled transporters by treating cells with phloridzin and furosemide and measuring ROS; 0.25 mM phloridzin inhibited ROS increase in MAP17-expressing clones (Figure 6A). Same results were obtained with 50 µM furosemide. Since the enhancement of tumorigenic properties is dependent on ROS, we tested whether phloridzin and furosemide also reduced tumorigenic capabilities in A375 melanoma cells expressing MAP17. To that end, we tested proliferation, clonability and growth in soft agar in the presence of both drugs. We found that the treatment with phloridzin and furosemide reduced the oncogenic properties of the MAP17-expressing cells: they have reduced growth rate at least during the first 4 days in culture. Later, they seem to adapt and recover full growing capabilities (Figure 6B). Colony formation efficiency in the presence of inhibitors also was reduced (Figure 6C). The same effect was found in the soft agar growth assay: phloridzin reduced the number and size of colonies of MAP17-expressing cells (Figure 6D and E).


Figure 6
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  		Fig. 6. Inhibition of Na+-coupled transporters inhibit MAP17-dependent ROS increase and tumorigenesis. (A) Phloridzin and furosemide inhibit ROS increase. To visualize intracellular ROS levels, A375 parental or MAP17-expressing cells were grown on cover slips, and then incubated at 37°C with 8 µM of CM-H2DCFDA as described in Experimental procedures. Intracellular ROS were visualized using a confocal ultra-spectral microscope Leica TCS-SP2-AOBS-UV. A representative picture of treated MAP17-expressing clones and parental cells is shown. (B) Phloridzin and furosemide inhibit growth rate. A growth curve of both parental cells (cells carrying empty vector, P) and MAP17-expressing clones (C3 and C4) was performed following the procedure described in Experimental procedures. Cells were treated with 50 µM furosemide or 0.25 mM phloridzin or excipient only as indicated. Data presented are the mean from triplicate samples; bars, ±SD. (C) MAP17 enhances cell survival through Na+-coupled glucose transporters. Triplicate cell samples were cultured for 10 days in the presence or absence of 50 µM furosemide or 0.25 mM phloridzin or excipient only (control), and then fixed and stained with crystal violet following the protocol described in Experimental procedures. Number of colonies counted. Parental cells (cells carrying empty vector, P) and MAP17-expressing clones (C3 and C4) were used in this experiment. Data presented are the mean from triplicate samples; bars, ±SD. (D and E) MAP17 increased colony number and size in soft agar is inhibited by Na+-coupled glucose transporters. Cells were grown in the presence of 50 µM furosemide or 0.25 mM phloridzin or excipient only (control), for 4 weeks then fixed, number of colonies counted. Presented data are the mean from three independent experiments; bars, ±SD. (E) Colony size was determined by measuring a minimum of 30 representative colonies. Parental cells (cells carrying empty vector, P) and MAP17-expressing clones (C3 and C4) were used in this experiment. Data presented are the mean from triplicate samples; bars, ±SD.

 
Therefore, inhibition of Na+-coupled co-transporters inhibits MAP17-dependent ROS increase and tumorigenesis.


   Discussion
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 Abstract
 Introduction
 Experimental procedures
 Results
 Discussion
Funding
 References
 
Melanoma cells that stably express MAP17 show an increased tumoral phenotype, with enhanced proliferative capabilities both in presence or absence of cell contact, decreased apoptotic sensitivity and increased migration. Moreover, we found that MAP17 is over-expressed, mostly through mRNA amplification, in a great variety of human carcinomas (14). The increased tumorigenic potential observed with the expression of MAP17 cDNA is in accordance with the increased expression of MAP17 observed in tumors, specially in ovarian and prostate tumors, where MAP17 levels are associated with malignant stages (14).

An initial report (6) described that over-expression in human carcinoma cells HT29 lead to a decrease in growth. We do not know the discrepancy between our data and those from Kocher et al. (6). It is possible that HT29 carry high internal ROS levels and a further increase induced by MAP17 reduced HT29 viability. We have over-expressed MAP17 into A375 and we have observed an increase of the tumorigenic properties. The same enhancement of tumorigenic properties have been obtained in at least three different carcinoma cell lines tested in other studies (data not shown).

The increased tumorigenic properties induced by MAP17 are associated with an increase in ROS since MAP17 increases endogenous ROS and the antioxidant treatment of MAP17-expressing cells entails a reduction in the tumorigenic properties of these cells. Two explanations can be offered for the mechanism by which ROS induce the transformed phenotype. First, reactive oxygen generated in the presence of MAP17 may be mutagenic, causing the transformed phenotype through the induction of mutations in oncogenes or tumor suppressor genes. Alternatively, ROS generated in a MAP17-dependent manner might function as an intracellular signal, inducing a growth-related genetic program. We have found that ROS removal by antioxidant treatments decrease the malignant cell behavior induced by MAP17, thus favoring the second hypothesis. Accumulating evidence implicates ROS in signaling cascades related to cell proliferation and transformation (18,20,21). Ras-transformed fibroblasts overproduce ROS, and this overproduction is correlated with the activation of mitogenic signaling pathways (18). Loss of Superoxidedismutase, SOD, (which should elevate ROS levels) has also been correlated with a tumoral phenotype and over-expression of SOD leads to the reversion of the transformed phenotype (22–24). On the other hand, H2O2 is generated in response to the growth factors EGF and PDGF, and is linked to growth-related signaling (20,25). When over-expressed in NIH3T3 mouse fibroblasts, Nox1, a NADPH oxidase catalytic subunit, induces excessive production of ROS and a transformed phenotype with increased mitotic rates and aggressive tumor formation in athymic mice (26). The phenotype of Nox1-transfected cells can be reversed by ROS reduction through stable expression of catalase, thereby implicating ROS as a signaling molecule (26).

The cellular targets for ROS signaling which are relative to growth and transformation are not well known. p42/p44 mitogen-activated protein kinase, p38 mitogen-activated protein kinase, p70S6k, signal transducers and activators of transcription, Akt/protein kinase B and phospholipase D-signaling pathways are all activated by ROS (25,27–29) but, in some cases, activation is indirect (30,31). A direct effect has been shown on protein tyrosine phosphatase-1B that is inhibited by oxidation of a thiol in the active site (32,33), leading to increased phosphotyrosines on many cell proteins. A variety of other targets can also be affected by ROS, including transcription factors such as nuclear factor-{kappa}B (34), activator protein-1 (35), PTEN (36) and p53 (37). ROS can directly modify signaling proteins through different modifications such as nitrosylation, carbonylation, disulphide bond formation and glutathionylation (38). Whatever the proximal targets, ROS reprogram the expression of enzymes and other proteins in the cell (39,40). DNA microarray experiments (26) indicate that up to 2% of the 6 500 genes queried are regulated by ROS. Furthermore, we have found that ROS increase activates PI3K pathway, probably by direct oxidation and inactivation of PTEN and other AKT phosphatases, maintaining AKT activated even in the absence of PI3K signal (Guijarro MV. et al. submitted for publication). AKT pathway activation induced by MAP17 expression might explain some of the properties described here. However, we think that other pathways must coexist induced by MAP17 at transcriptional level, as described in other systems (39,40).

But, how are ROS increased by MAP17? One possibility is that MAP17 can increase glucose and mannose uptake, inducing an increase in metabolism and ROS as side product. However, the enforced increase of the intracellular mannose or glucose level without MAP17 over-expression does not alter the tumorigenic properties of the cells (data not shown). We therefore favor a hypothesis in which ROS are generated with the direct participation of MAP17.

It has been proposed that the effect of MAP17 most probably consists in an enhancement of the endogenous uphill transport system (11). MAP17 also presents a weak similarity with ATP synthases, Na-solute transporters and transferases. However, both the structural simplicity of MAP17 and the kinetic analysis of the induced transport (11) suggest that MAP17 is an activator of the capacity of an endogenous transporter. We suggest that MAP17 enhances the activity of membrane transporters, thus inducing an increase in intracellular ROS. Subsequently, this increase in ROS causes the increase in the malignant behavior of tumoral cells. We have tested this hypothesis using Na+-coupled transporter inhibitors. These inhibitors block MAP17-dependent ROS increase in A375 cells and subsequent encancement of malignant behavior, confirming the role of membrane transporters in MAP17 effect. MAP17 could modulate the activity or the organization of membrane transport through direct interaction as the RS1 modifier does (41) or through competition for PDZ-binding places to alter the stoichiometry of the transporter–PDZ proteins (11). The structural simplicity of MAP17 supports this regulatory role.

In summary, in this report, we show that MAP17 enhances the tumorigenic capabilities of the cells through an increase in intracellular ROS. Therefore, MAP17 confers selective advantage to cells growing in the tumor allowing the selection of clones that over-express this protein. Our data point out a new type of oncogenic proteins and remark the role of ROS as signaling molecule in tumorigenesis. Furthermore, we suggest that MAP17-increased tumorigenesis is dependent upon Na+-coupled transport-dependent ROS increase.


   Funding
Top
 Abstract
 Introduction
 Experimental procedures
 Results
 Discussion
 Funding
References
 
Spanish Ministry of Health (FIS-02/0126); Fundacion Mutua Madrileña and Spanish Ministry of Education and Science (SAF2005-00944).


   Acknowledgments
 
The authors acknowledge the other members of the Assay Development Group at Spanish Centro Nacional de Investigaciones Oncológicas and Jim Bischoff for helpful discussions and critical reading of the manuscript. We also thank the Spanish National Cancer Tumor Bank Network that kindly provided the prostate tumor samples.

Conflicts of interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Experimental procedures
 Results
 Discussion
 Funding
 References
 

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Received March 27, 2007; revised May 8, 2007; accepted May 14, 2007.


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