Introduction
The gene encoding Na
+/H
+ exchanger regulatory factor 1 (NHERF1) (also known as EBP-50 or NHERF) is a candidate tumor suppressor gene in human breast cancer. Human
NHERF1 cDNA encodes a protein of 358 amino acids in length. NHERF1 and its close homolog NHERF2 (also known as E3KARP or TKA1) share two modular structures: two tandem PDZ domains at the amino-terminus and an ezrin-radixin-moesin (ERM)-interacting domain at the carboxyl-terminus [
1]. NHERF1 and NHERF2 are differentially expressed in mammalian tissues, with particularly high levels found in polarized epithelial cells [
2]. NHERF1 acts as an important regulator and integrator of multiple signaling pathways by virtue of its ability to bind to a variety of proteins through its PDZ (PSD-95/Dlg/ZO1) domains and ERM-interacting domain. Via its PDZ domains, NHERF1 recognizes a carboxyl-terminal motif, D(S/T)XL, that is present in a number of transmembrane proteins, such as platelet-derived growth factor receptor (PDGFR) [
3,
4], cystic fibrosis transmembrane conductance regulator [
5‐
7], β
2-adrenergic receptor [
8,
9], and sodium bicarbonate co-transporter [
10]. NHERF1 also interacts with a variety of intracellular proteins, including phospholipase C-β isoforms, G protein-coupled receptor kinase 6A, spleen tyrosine kinase (SYK) and Yes-associated protein 65 [
11‐
15]. Via its carboxyl-terminal ERM-interacting domain, NHERF1 binds to ERM proteins, a family of actin cytoskeletal adaptors. One ERM family member is merlin, the product of
NF2 tumor suppressor gene. Germline mutations of
NF2 have been implicated in predisposition to meningiomas and schwanomas [
16,
17]. The amino-terminus of the ERM family proteins (ERM domain) binds to the ERM-interacting domain of NHERF1; this interaction may be important for NHERF1 functions through its connection of membrane transporters and actin cytoskeleton.
The
NHERF1 gene is located at 17q25.1. Loss of heterozygosity (LOH) at this locus occurs in more than 50% of breast tumors [
15,
18‐
23]. However, such allelic loss is infrequent in other tumor types; LOH at the
NHERF1 locus occurred in fewer than 10% of colorectal and pancreatic cancer lines, suggesting that
NHERF1 is specifically targeted during breast tumorigenesis [
15]. We reported three cases of
NHERF1 intragenic mutations in a panel of breast tumors pre-screened for LOH [
15]; notably, all mutations were located at conserved residues of PDZ domains or ERM-interacting domain. These tumorigenic mutations interfere with NHERF1 binding to SYK or merlin (loss-of-function), suggesting their functional relevance to breast tumor initiation or progression.
NHERF1 LOH positively correlates with aggressive features of breast tumors, including tumor size, grade and stage, indicating that
NHERF1 plays a critical role in mammary carcinogenesis, in which its putative suppressor activity may be haplo-insufficient.
Despite the genetic evidence available, the biologic activities of NHERF1 in mammary gland were unknown. The finding that the phosphorylation status of NHERF1 oscillated during the cell cycle implicated possible link of NHERF1 to tumor-related response [
24]. Indeed, the fact that NHERF1 is a substrate for cdc2, a G
2 to M phase cyclin-dependent kinase, suggests a role of NHERF1 in cell division [
24]. Using the small interfering RNA (siRNA) method, we demonstrated increased growth of breast cancer cells when NHERF1 expression was knocked down [
25]. The growth promotion effect that occurred in response to NHERF1 loss was due to an accelerated G
1 to S transition, which was accompanied by elevated levels of cyclin E and phosphorylated Rb protein. This indicated that normal NHERF1 function may involve suppression of cell cycle progression [
25].
Although the identity of the NHERF1-interacting partner responsible for the cell cycle regulatory effect remains unclear, a recent report [
26] showed NHERF1 binding to the carboxyl-terminal tail (PDZ-binding motif) of phosphatase and tensin homolog (PTEN). The PDZ-binding motif of PTEN was also shown to interact with membrane-associated guanylate kinase family proteins, but the biologic significance of these bindings is not clear [
27‐
29]. The interaction of NHERF1 with PTEN and PDGFR facilitates the formation of a ternary complex. Interestingly, this complex formation was found to offset PDGF-initiated phosphorylation of downstream targets such as Akt in mouse embryonic fibroblasts (MEFs) [
26]. Activated Akt (by phosphorylation) plays a pivotal role in promoting cell survival, increasing cell invasiveness and overriding cell cycle checkpoints [
30]. A possible activity of NHERF1 in counteracting the Akt pro-oncogenic pathway raises an attractive mechanism that explains NHERF1 tumor suppressor activity in mammary glands. Thus, in the present study we sought to determine whether the activity of NHERF1 is associated with a PTEN-dependent pathway in breast cells, and whether NHERF1 expressional status affects PDGF-stimulated downstream cell survival signaling as well as cell responses to PDGFR inhibition.
Materials and methods
Cell culture
Breast cancer cell lines MCF7, MDA-MB-468, SKBr3, T47D, ZR75.1 and immortalized mammary epithelial line MCF10A were purchased from American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in recommended media.
Cultured Zr75.1, MCF10A and MEF cells were incubated with serum-free media for 1 day. They were then treated with PDGF-BB (0.5 ng/ml; Millipore, Billerica, MA, USA) for 0 to 120 minutes before cells were harvested in 1× SDS sample buffer.
NHERF1knockout mice
NHERF1+/- mice [
31] were inbred to generate littermates of three genotypes. Duplex PCR was used to genotype
NHERF1 knockout mice. A common forward primer (5'-ctctgtttattcccagaagga-3') was included in the PCR reaction, together with reverse primers for knockout (5'-caagctcttcagcaatatcac-3') and wild-type (5'-ggttctaccagacggataaac-3') genotypes that were expected to yield 2.4-kilobase and 1.4-kilobase products, respectively. PCR conditions were described previously [
15].
To harvest mammary gland of NHERF1 knockout mice, 10-week-old female littermates were killed by carbon dioxide inhalation. Mammary glands were collected and snap frozen at -80°C until use. Tissues were then ground while frozen in liquid nitrogen and lysed in 1× SDS sample buffer. The lysates were then subjected to immunoblotting.
To obtain MEF cells of varied NHERF1 genetic backgrounds, the NHERF1+/- mice were inbred. At embryonic days 12 to 14, the embryos were dissected to remove heads and internal organs. The remaining tissues were minced, trypsinized and plated in Dulbecco's medified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Genomic DNA was also harvested from MEF cells for genotyping. Early-passage MEF cells were then subjected to experimental treatment.
DNA constructs
To prepare recombinant retrovirus to knockdown NHERF1 expression, an siRNA sequence that targeted NHERF1 transcript was inserted into pBabe-U6-Puro (a gift from Dr Jinsong Liu, M.D. Anderson Cancer Center, Houston, TX, USA). Two oligonucleotides (5'-ggaaactgacgagttcttcaaagctttgaagaactcgtcagtttccctttttg-3' and 5'-aattcaaaaagggaaactgacgagttcttcaaagctttgaagaactcgtcagtttcc-3') were annealed and subcloned into pBabe-U6, creating pBabe-U6/NHERF-910-Puro. Similarly, oligonuclotides (5'-ggacgaactggtgtaatgatatgaagcttcatatcattacaccagttcgtccctttttg-3' and 5'-aattcaaaaagggacgaactggtgtaatgatatgaagcttcatatcattacaccagttcgtcc-3') were used to knock down PTEN expression (pBabe-U6/PTEN-Puro).
GST-PTEN, pcDNA3.1-PTEN, and pcDNA3.1-PTEN-C124S were gifts from Dr Charles Sawyer (University of California, Los Angeles, CA, USA). To make a PTEN construct with deletion of the last six residues (TQITKV), PCR was conducted to introduce premature termination, using pcDNA3.1-PTEN as a template. This procedure created pcDNA3.1-PTEN-ΔC.
NHERF1 cDNA was obtained by reverse transcription from mRNA of a breast cancer cell line, SKBr3, and amplified by PCR. Reverse transcription PCR products were subcloned into a TA cloning vector (Invitrogen). NHERF1 cDNA was then subcloned into pcDNA3.1(+) (Invitrogen), generating pDNA3.1-NHERF1. NHERF1 cDNA was then enzymatically released from pcDNA3.1-NHERF1 and subcloned into pBabe-Puro, pBabe-Neo (Addgene, Cambridge, MA, USA), and pGEX2TK (GE Healthcare, Piscataway, NJ, USA), yielding pBabe-NHERF1-Puro, pBabe-NHERF1-Neo, and pGEX2TK-NHERF1, respectively. cDNA fragments encoding PDZ-I (residues 1 to 150), PDZ-II (residues 97 to 239), PDZ-I&II (residues 1 to 239), and carboxyl-terminus (residues 231–358) of NHERF1 were PCR-amplified and constructed to pcDNA3.1 or pGEX2TK. All constructs generated by PCR were verified by automated DNA sequencing.
Retroviral infection
Retroviral stocks were made by transfecting packaging cells (amphotropic Phoenix cells) with retroviral constructs (pBabe-U6-Puro, pBabe-U6-NHERF-910-Puro, pBabe-U6-PTEN-Puro, pBabe-Puro, pBabe-NHERF1-Puro, pBabe-Neo, and pBabe-NHERF1-Neo), using Fugene 6 (Roche Applied Science, Indianapolis, IN, USA).
To prepare MCF10A cells over-expressing NHERF1, MCF10A cells were infected with NHERF1-Puro (or NHERF1-Neo for PTEN knockdown cells) recombinant retrovirus. Infected cells were selected by adding 1 to 5 μg/ml puromycin (or 600 μg/ml G418) to culture media. Surviving cells were assessed for NHERF1 expression by immunoblotting. MCF10A-NHERF1-Neo cells were further infected with PTEN-siRNA retrovirus to knockdown PTEN expression, which was determined by PTEN immunoblotting.
The expression of NHERF1 in Zr75.1 and MDA-MB-468 cells was knocked down by using retrovirus-based siRNA method. Zr75.1 cells were infected with Babe-U6-NHERF-910-Puro retrovirus and subjected to puromycin (2 μg/ml) selection. NHERF1 expression was determined by immunoblotting.
Glutathione S-transferase pull-down assays
Glutathione S-transferase (GST) pull-down assays were used to assess the interaction of NHERF1 and PTEN. GST-PTEN or GST-NHERF1 and their derivatives were induced by isopropyl-β-D-1-thiogalactopyranoside in pGEX2TK-transformed BL21 strain and purified with glutathione-Sepharose beads (GE Healthcare).
Radio-labeled full-length or defined segments of NHERF1 and full-length or truncated PTEN were synthesized by in vitro transcription from pcDNA3.1 plasmids containing the NHERF1 or PTEN cDNA and translated in the presence of [35S]methionine (T7 Quick TNT kit; Promega, Madison, WI, USA). The translation products were mixed with purified GST-PTEN or GST-NHERF1 immobilized on the beads. Pull-down assays were performed at 4°C for 1 hour in 1× binding buffer (20 mmol/l Tris [pH 7.5], 150 mmol/l NaCl, and 1% NP-40). The beads were then washed thoroughly with 1× binding buffer. Bound proteins were eluted by boiling in 1× SDS sample buffer, separated by SDS-PAGE. Ten per cent of the input TNT lysates was also run on the PAGE to determine relative binding capacity. The PAGE gel was then dried and exposed for autoradiography.
GST pull-down assay was also used to determine whether endogenous PTEN or NHERF1 binds to recombinant NHERF1 or PTEN, respectively. MDA-MB-468, MCF7, and T47D cells were harvested by adding 1× lysis buffer (50 mmol/l Tris-HCl [pH 7.5], 150 mmol/l NaCl, 1 mmol/l EDTA, 1% Triton-X 100, 10 mmol/l NaF, 1 mmol/l Na3VO4 and 1× protease inhibitor cocktail [Sigma, St. Louis, MO, USA]). The cell lysates were then incubated with beads coated with GST fusion proteins. The incubation lasted for 2 hours at 4°C. The beads were then washed thoroughly with 1× binding buffer before being boiled in 1× SDS sample buffer. The eluted proteins were then subjected to NHERF1 or PTEN immunoblotting.
Immunoprecipitation and immunoblotting
To assess the interaction of PTEN with NHERF1 at the endogenous level, cultured MCF7 or Zr75.1 cells were lysed with 1× NETN buffer (20 mmol/l Tris-HCl [pH 7.5], 150 mmol/l NaCl, 1 mmol/l EDTA, 0.5% NP-40, 30 μg/ml aprotinin, 5 mmol/l PMSF, 25 mmol/l NaF, and 2 mmol/l Na3VO4). The soluble proteins (about 600 μg) were immunoprecipitated with 2 μg of goat IgG reactive to PTEN (N-19; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) at 4°C overnight, using normal goat IgG as a control. The immunocomplex was collected by addition of 50 μl of agarose-conjugated protein G (Roche Applied Science) and detected with anti-NHERF1 antibody.
Protein concentrations were measured by using BCA reagent. Lysates were then subjected to immunodetection of phospho-Akt (p-Akt) and total Akt. Immunoblottings were carried out essentially as described previously [
32]. Antibodies used were human NHERF1 (EXBIO Praha, Vestec, Czech Republic), β-actin (Santa Cruz Biotechnology), mouse NHERF1 (Affinity Bioreagents, Golden, CO, USA), PTEN (Millipore), caspase-3, phospho-Akt-Ser473, total Akt, and phospho-p70 S6 kinase-Thr421/Ser424 (Cell Signaling Technology, Danvers, MA, USA).
MTT assays
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays were used to measure cell viability. Cells were seeded in 96-well cluster dishes at 5,000 cells/well with 100 μl complete medium. After overnight incubation, medium was replaced to include various concentrations of STI-571 (Novartis Pharmaceuticals). After 2 days of treatment, cells were fed 100 μl fresh medium that contained 1 mg/ml MTT (Sigma). The incubation lasted for 2 hours before the medium was removed and cells dissolved in 150 μl dimethyl sulfoxide. Absorbance was measured using a multiSkan plate reader (LabSystems, Waltham, MA, USA) at a wavelength of 570 nm. Each sample was processed in triplicate. Experiments were repeated at least three times.
Discussion
Our earlier results of
NHERF1genetic alterations in human breast cancer prompted us to hypothesize that
NHERF1 acts as a tumor suppressor gene in mammary gland [
15]. Human
NHERF1 is an estrogen-inducible gene [
36]. Estrogen response element half-sites have been located in the 5'-regulatory region of
NHERF1 gene that are responsible for estrogen-stimulated expression [
38]. Because estrogenic signaling is thought to be growth promoting in breast cancer [
39], the suppressor activity of NHERF1 was somewhat unexpected. Although the roles of estrogenic induction in NHERF1 biology is not clear, we identified increased growth of breast cancer cells after NHERF1 knockdown [
25], which is in agreement with the proposed tumor suppressor function of
NHERF1. Here we provide additional evidence to support our overall hypothesis.
In breast cells, NHERF1 expression accelerated the turnover of p-Akt induced by PDGF stimulation. This finding was obtained in both over-expression and knockdown models, from both immortalized normal mammary epithelial cells (MCF10A) and a breast cancer line (Zr75.1). The effect of NHERF1 to stimulate the decay of p-Akt is probably through PTEN recruitment by NHERF1 to the cytoplasmic membrane compartment, where active phosphorylation and dephosphorylation of Akt occur. The response is probably related to normal mammary biology, as indicated by the markedly increased levels of p-Akt in the mammary gland tissues of
NHERF1-/- mice (Figure
4). Elevated p-Akt resulting from
NHERF1 deletion presumably potentiates the cell survival pathway in mammary gland, where balanced survival and apoptotic signaling is essential for normal development and homeostasis [
40]. A deregulated apoptotic process, which leads to defective structural organization and remodeling in mammary gland, is believed to be directly related to breast cancer etiology [
41,
42]. Interestingly, we recently found that, in contrast to the wild-type mice,
NHERF1-/- mice exhibited increased ductal side branching and extensive alveolar hyperplasia in mammary gland (our unpublished data). Whether aberrant Akt activation is directly related to hyperplastic morphology in mammary gland warrants further investigation. Also remaining to be established is whether this impaired mammary development is sufficient to increase or accelerate the incidence of mammary tumor.
Because of the strong correlation between
NHERF1 LOH and the aggressive features of breast cancer, we hypothesized that NHERF1 tumor suppressor activity was haploinsufficient [
15]. A number of tumor suppressor genes have been shown to have haploinsufficient activities [
43‐
46]. In these cases, mice carrying an inactivated allele are predisposed to tumor development, and the resulting tumors frequently retain a functional wild-type allele. Interestingly, two NHERF1-binding partners, namely PTEN and NF2, use this mechanism [
43,
44]. A lowered expression as a result of single allele deletion of the
NHERF1 gene is obligatory for its haploinsufficient biology. In support of this mechanism, we found that LOH of
NHERF1 locus in 22 breast cancer cell lines was strongly correlated with lowered NHERF1 protein level. The haploinsufficient expression of
NHERF1 is also supported by
in vivo observations. Monoallelic deletion of
NHERF1 was shown to decrease NHERF1 expression in kidney epithelial cells [
31]. Similarly, deletion of one allele of the
NHERF1 gene resulted in decreased NHERF1 protein expression in mammary gland (Figure
4), providing a plausible link between altered protein expression level and the resultant phenotypic responses. We found lowered NHERF1 expression to be accompanied by a modest increase in p-Akt in the mammary tissue of
NHERF1+/- mice as compared with that in wild-type (Figure
4). Coincidentally, the mammary glands of
NHERF1+/- mice exhibited alveolar hyperplasia, albeit less extensively than the
NHERF1-/- mice (our unpublished data). The correlation suggested an association between abnormal Akt activation and mammary hyperplasia, highlighting the biochemical and pathological consequences of monoallelic deletion of
NHERF1.
Our findings clearly indicated a dosage effect of NHERF1 activity during normal mammary gland development. Whether NHERF1 affects breast cancer susceptibility through the haploinsufficiency mechanism requires further investigation. Haploinsuficient NHERF1 tumor suppressor activity would also explain the relatively low frequency of intragenic mutations, although the possibility that other NHERF1 pathway components are genetically altered cannot be ruled out.
The present study verifies that NHERF1 has tumor suppressor activity, providing evidence to suggest that its function relies on an intact PTEN pathway. First, NHERF1 is associated with accelerated dephosphorylation of p-Akt, presumably through recruitment of PTEN by NHERF1. Second, knockdown of PTEN abolishes NHERF1-induced sensitivity to chemo-agents. If NHERF1 activity is dependent on PTEN, as our functional study had suggested, then intact NHERF1 should be associated with altered PTEN (or PI3KCA) gene in breast cancer. Our data from 39 breast cancer cell lines showed that this was indeed the case. Collectively, our present study indicates that NHERF1 binds to PTEN to downregulate the PI3K-Akt pathway to elicit tumor suppressor activity. Given that PTEN-PI3K-Akt is one of the most prominent pathways relevant to tumorigenesis and targeted therapy of almost all types of carcinoma, studies on NHERF1 should be instrumental to the development of new strategies to overcome chemo-resistance and enhance efficacy.
In this study we also present evidence that NHERF1 expression status significantly affects how cells respond to PDGFR inhibition. PDGF is among the key growth factors and cytokines that breast cancer cells produce via autocrine or paracrine mechanisms that contribute to malignant progression [
47]. Activated PDGF signaling has been shown to prevent cells from undergoing apoptosis during epithelial mesenchymal transition and thus promote breast cancer progression and metastasis [
48,
49]. As a potent PDGFR inhibitor, STI-571 has been shown to inhibit breast cancer bone metastasis in mouse models [
50], and it is being tested clinically in treatment of metastatic breast cancer, among other cancer types [
51]. Although the exact mechanism responsible for improved susceptibility to STI-571 by NHERF1 needs further investigation, our present study indicates an inhibitory effect of NHERF1 on PDGF-medicated breast cancer progression and suggests that the status of NHERF1 expression in breast tumor influences how patients respond to STI-571. Because a majority of breast tumors lose NHERF1 expression, our present study raises a possibility of enhancing chemosensitivity by restoring NHERF1 expression. NHERF1 expression may also be used as a biomarker to predict the effectiveness of such treatment.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YP designed the experiments, carried out technical procedures, and interpreted the data. EJW provided the knockout animals and edited the manuscript. JLD designed the experiments, interpreted the results and wrote the manuscript. All authors approved the final manuscript.