Autocrine Motility Factor Promotes HER2 Cleavage and Signaling in Breast Cancer Cells Free

HER2 (ERBB2/Neu), a family member of EGF receptors (HER) is overexpressed in approximately 25% of invasive breast carcinomas (1–3) and is a major approved target for breast cancer therapy. The crystal structure of HER2 suggests that its extracellular domain (ECD) exists in a constitutively active conformation resembling the ligand-bound state of the other HERs (4, 5), whereas HER2-ECD–targeting antibodies that are antagonistic or agonistic at the levels of HER2 phosphorylation and cell growth, suggest the presence of binding partner(s) necessary for complete activation of HER2 (1, 6, 7). Herceptin/trastuzumab has improved the outcome in patients with HER2-overexpressing breast carcinoma (8, 9). However, a substantial proportion of patients with HER2-positive breast cancer is intrinsically resistant to trastuzumab or acquires resistance following initial treatment (10). The mechanisms of resistance to Herceptin/trastuzumab are mainly involved in the restoration of the PI3K/AKT signaling pathways either via an epitope masking (Mucin) and escaping (truncated p95HER2), alternative compensation of receptor tyrosine kinases, or the constitutive mutations of phosphoinositide-3-kinase (PI3K) pathways (10–12). Retrospective studies suggest that the oncogenic p95HER2 variant is most likely responsible for clinical resistance to Herceptin/trastuzumab treatment (13, 14).

Phosphoglucose isomerase (EC: 5.3.1.9; PGI) is a housekeeping dimeric enzyme that catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate in glycolysis/gluconeogenesis (15). PGI belongs to the moonlighting family of proteins having multiple functions/activities within a single polypeptide chain, not resulting from multiple domains of a protein, alternative RNA splicing, gene fusions, and/or posttranslational processing (16). Secreted form of PGI in the extracellular milieu of transformed cells and several tissues was identified as neuroleukin (NLK), a neurotrophic factor that mediates the differentiation of neurons and autocrine motility factor (AMF), a tumor-secreted C–X–X–C cytokine that is involved in cell motility (17, 18). Aberrant secretion of AMF was observed in the blood and urine of patients with cancer, suggesting a prognostic value (15, 19). Functionally, AMF was shown to induce cell proliferation, differentiation, and survival of various cancer and immune cells (15). Independent reports have shown that AMF activates mitogenic mitogen-activated protein kinase (MAPK)/ERK or prosurvival PI3K/AKT pathways, similarly to the signaling mode of growth factors as emphasized in the resistance to HER2-targeted therapy (20, 21). The receptor of AMF, that is, gp78/AMFR was identified as a 7 transmembrane domain containing protein. However, gp78/AMFR-null cells still respond to AMF, suggesting the presence of yet another unidentified receptor (22, 23).

Here, we show that in human breast carcinoma cells AMF binds to HER2, induces its phosphorylation, ectodomain shedding, activates its downstream signaling pathways, and overcomes Heceptin/trastuzumab effect. The data suggest that AMF may be a novel therapeutic target for patients with breast cancer in conjunction with Heceptin/trastuzumab therapy.

Purified rabbit PGI/AMF was purchased from Sigma for AMF stimulation. Monoclonal anti-PGI (12F9A6, Pfizer) and rabbit anti-PGI (H300, Santa Cruz) antibodies were used for Western blot analysis and immunoprecipitation. p-ERK (E-4), ERK1/2(MK1), p-Tyr (PY20), anti-HER2-ICD (Neu, C-18), anti-HER2-ECD (9G6), p-HER2 antibodies, and lapatinib were purchased from Santa Cruz. Anti-p-AKT (Ser473) and AKT antibodies were from Cell Signaling. Anti-rabbit IgG-TRITC and anti-IgG-FITC antibodies, Marimastat (BB2516), lysophophatidic acid, and pertussis toxin (P2980) were purchased from Sigma. Wortmannin and U0126 were obtained from Calbiochem. 3,3′-Dithiobis(sulfosuccinimidylpropionate) [DTSSP] was purchased from Pierce. Trastuzumab was a kind gift from Dr. Wei-Zen Wei of Wayne State University (Detroit, MI). Anti-V5, anti-HER2-ECD antibodies (poly-2 and CB11 clone), siRNAs against gp78, HER2, and AMF were purchased from Invitrogen. MTT was purchased from Sigma.

T47D and EBNA 293 cells obtained from American Type Culture Collection were grown in Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and antibiotics. SkBr3 and BT474 were kindly gifted by Dr. Arun Rishi of Wayne State University. SkBr3 cells were cultured in complete McCoy's modified 5A medium. Before pretreatment with inhibitors or addition of stimulators (EGF, AMF), 50% confluent cells were rinsed 2 times with 1× PBS and then serum-starved for 16 hours.

Cross-linking with DTSSP was conducted to identify interaction of AMF (AMF-V5) and HER2. T47D cells were washed with 1× PBS and then exposed to AMF (AMF-V5) along with DTSSP for 1 hour at 4°C. Reactions were terminated by the addition of 20 mmol/L Tris–HCl (pH 7.5) for 15 minutes at room temperature. Cells were extracted with lysis buffer and insoluble material was removed by centrifugation and supernatants were processed for immunoprecipitation.

Expression plasmid of AMF-V5 was constructed in pCDNA3.1/V5 (Invitrogen) harboring human full-length PGI/AMF and confirmed by sequence analysis. CMV-HER2 (p185HER2) was purchased from Addgene and transiently transfected into EBNA293 cells using Fugene HD (Roche). Breast cancer cells were seeded at 50% confluence per well in 6-well plates overnight and transfected for 48 hours with 20 nmol/L siRNAs using Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer's instructions.

Western blotting and immunoprecipitation was conducted according to routine protocol. Briefly, the cells were extracted in lysis buffer [20 mmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% NP-40, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na₃VO₄, and 1 μg/mL leupeptin]. After BCA protein assay (Pierce), immunoprecipitation with appropriate antibodies (1 μg) was carried out overnight. Each experiment was repeated at least, twice.

For immunofluorescence studies, SkBr3 cells grown on 12-mm round coverslips (Fisher) were processed after AMF-V5 treatment for 15 minutes. The cells were washed and fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.2% Triton X-100 for 5 minutes. For double immunofluorescence labeling, the cells were incubated with primary antibodies (anti-HER2 and anti-V5) for 1 hour separately to minimize the cross-reactivity. After 3 washes, appropriate secondary antibodies (fluorescein isothiocyanate [FITC]-conjugated, tetramethylrhodamine isothiocyanate [TRITC]-conjugated) were also incubated separately. Immunofluorescence images were obtained using a Zeiss Confocal Laser Microscope LSM 510. Four micrometer sections obtained from formalin fixed, paraffin-embedded tissue blocks were deparaffinized, rehydrated, and microwaved on high 2× for 5 minutes in 1 mmol/L sodium citrate buffer, pH 6.0. The sections were washed 3× in PBS and blocked with Super Block (Skytek Laboratories) for 10 minutes. Sequential sections were then incubated in PBS and linked with biotinylated mouse anti-AMF antibodies at 4°C overnight. The sections were then washed 3× for 10 minutes each in PBS and linked with biotinylated anti-mouse secondary antibodies (Vector Laboratories) followed by Texas Red–conjugated Avidin. After washing, the sections were incubated at 4°C overnight with rabbit anti-HER2 antibodies (Invitrogen) and with fluorescein-conjugated anti-rabbit secondary antibodies. Images were documented with an OLYMPUS BX40 microscope supported by a DP72 CCD Camera, and CellSens Dimension Imaging Software (Olympus).

BT474 cells were seeded at a density of 5 × 10⁴ cells per well in 24-well plates. After 24 hours, cells were treated with either 40 μg/mL trastuzumab or a combination of AMF (90 nmol/L) with trastuzumab. Cell growth was monitored at 550 nm after MTT assay. All experiments were carried out in quadruplicates and repeated twice. Statistical analysis was done using Student t test. P < 0.005 was regarded as significant.

Transwell (Corning Costar) migration assays were conducted on SkBr3 cells following pretreatment with trastuzumab for 16 hours. A total of 5 × 10⁴ SkBr3 cells in serum-free medium were introduced into the upper compartment of Transwell chambers (8 μmol/L pore) and were allowed to migrate in lower chambers with 10% FBS or AMF (0.1 μg/mL) for 16 hours. Migrated cells were fixed and stained with Hema-3 stain kit (Fisher). Each condition was assayed in triplicate and each experiment was repeated 3 times.

To study whether AMF acts independently of HER2 signaling, we initially established whether AMF induces PI3K/MAPK signaling pathways through gp78/AMFR in low HER2-expressing breast cancer cell line T47D. Treatment with purified AMF showed that it activates both PI3K/AKT and MAPK/ERK (Fig. 1A–C). Furthermore, gp78/AMFR knockdown study revealed that gp78/AMFR mediates AMF-induced p-ERK but not p-AKT, suggesting involvement of another AMF receptor for AKT activation (Supplementary Fig. S1). Because participation of a pertussis-toxin sensitive G protein in the signal transduction of AMF-stimulated cell motility was previously reported (24), we ventured that presence of yet another AMF receptor might be a G protein coupled receptor (GPCR), which transactivates the HER family through ectodomain shedding of membrane-bound HER ligand precursors by matrix metalloproteinases (MMP), resulting in the activation of PI3K/MAPK pathways (25). Therefore, we tested whether AMF is involved in the transactivation of the HER family for AKT activation. Interestingly, the results show that AMF-induced p-AKT/p-ERK was partially suppressed by lapatinib [dual kinase inhibitor of EGF receptor (EGFR) and HER2] compared with control and EGF, indicating that AMF-induced p-AKT/p-ERK is mediated by HER receptors and implying the responsibility of gp78/AMFR for partial suppression by lapatinib (Fig. 1D). To study whether a GPCR mediates AMF-induced p-AKT/p-ERK, we conducted suppression studies using pertussis-toxin (G protein inhibitor) or Marimastat (MMPs inhibitor) for blocking GPCR signaling. We found that these inhibitors did not suppress AMF-induced p-AKT/p-ERK (Fig. 1E), signifying that AMF signaling is not linked to GPCR but independently activates EGFR and/or HER2.

As HERs phosphorylation is induced by homo- and heterodimerization following the engagement of HER ligand and a cognate receptor (2), we addressed whether AMF induces the phosphorylation of EGFR and/or HER2. As shown in Fig. 2A, phosphorylation of both EGFR and HER2 were induced by exogenous EGF, which does not bind to HER2 (2, 3), implying EGFR/HER2 heterodimerization. While AMF did not induce phosphorylation of EGFR and kinetic studies indicated that AMF induces the phosphorylation of HER2 within a very short time frame of 2 minutes (Fig. 2B). Moreover, we found that both background p-HER2 and unphosphorylated p185HER2 vanished concomitantly with the emergence of cleaved p95HER2. As ectodomain shedding of p185HER2 was previously reported to generate membrane-anchored p95HER2 (95 to 100 kDa) by sheddases such as MMPs at the juxtamembrane region of HER2 (26), we addressed whether HER2 cleavage is directly affected by AMF or is mediated by MMPs. MMPs inhibitor suppressed AMF-induced cleavage of p185HER2 and also stabilized phosphorylated HER2 by AMF (Fig. 2C), indicating that AMF is sufficient for the phosphorylation of HER2 and AKT activation and also cooperates with MMPs for HER2 cleavage.

Parental EBNA293 cells did not show AKT/ERK activation after AMF treatment (Supplementary Fig. S2). To verify that AMF indeed triggers phosphorylation and participates in the cleavage of HER2, the p185HER2 expression vector was transfected into EBNA293 cells. The results showed that p-HER2 induction in response to AMF seemed together with cleaved p95HER2 (Fig. 2D) and AMF-induced HER2 cleavage was mediated by MMPs (Fig. 2E), consistent with the earlier results (Fig. 2B and C). We confirmed the existence of AMF/HER2/AKT axis by HER2 knockdown in T47D cells, in which AMF-induced AKT activation was partially suppressed (Fig. 2F).

Because the data indicate that AMF induces phosphorylation of HER2, we tested the interaction of exogenously added AMF and HER2, using a membrane impermeable cross-linker (DTSSP), which establishes extracellular association of proteins. Exogenous AMF was coimmunoprecipitated with HER2 (Fig. 3A, top). To further verify this interaction V5-tagged AMF (AMF-V5) was expressed and purified to rule out any possible background experimental noise from the endogenous AMF. The interaction of exogenous AMF-V5 with HER2 was clearly observed by reciprocal coimmunoprecipitation studies (Fig. 3A, middle and bottom). In addition, immunofluorescence studies revealed the colocalization of HER2 and exogenous AMF-V5 (Fig. 3B).

To elucidate whether AMF interacts with the ectodomain of HER2, HER2-transfected cells were extracellularly cross-linked with either exogenous AMF or AMF-V5. We observed that AMF or AMF-V5 cross-linked HER2 molecules were not immunoprecipitated with anti-HER2-ECD antibodies and immunoprecipitated HER2 levels were reduced compared with trastuzumab (juxtamembrane) or anti-HER2-ICD antibodies (Fig. 3C), suggesting that AMF–HER2–binding motif is either overlapping or close to the epitope of anti-HER2-ECD antibody. Next, taking advantage of the data obtained from the crystal structures of AMF and the ECD of HER2 (4, 27), we used the ZDOCK software (http://zdock.bu.edu) to predict their physical interactions. AMF binding to HER2 ectodomain was mapped to the molecular region of HER2 between domain I and III (Fig. 3D)

As HER2 overexpressing cells have a high expression level of endogenous p-HER2 via a ligand-independent mechanism (1–3), we examined how AMF is able to affect endogenous p-HER2 and HER2 in HER2-overexpressing cells. Also, we questioned whether AMF-induced downstream events might be blocked by anti-HER2-ECD antibodies, which can compete with AMF for binding to HER2. As shown in Fig. 4A, the treatment with anti-HER2-ECD antibodies alone showed that it induced p-HER2 levels in concert with HER2 cleavage and AKT/ERK activation, mimicking AMF actions. Combination of AMF and anti-HER2-ECD antibodies shows synergistic effect. Interestingly, HER2 shedding was induced by exogenous AMF regardless of the presence of Herceptin/trastuzumab in the conditioned media. AMF treatment overcame Herceptin/trastuzumab–mediated suppression of HER2 cleavage and p-AKT/p-ERK levels.

Next, as AMF-induced p95HER2 is an indicator of trastuzumab resistance through epitope escaping (10), we proceeded to validate the effect of AMF on cell growth and motility with trastuzumab treatment. We observed AMF-induced HER2 cleavage in cells treated with trastuzumab in complete medium (Fig. 4B). As a result, exogenous AMF partially overcame the inhibitory effect of trastuzumab on cell growth and motility (Fig. 4C and D).

Next, we examined the relationship between AMF and HER2 expression. AMF secretion was observed to be positively correlated with the expression of both p95HER2 and p185HER2 proteins (Supplementary Fig. S3). Sequentially, we found that HER2 knockdown inhibits AMF secretion, suggesting that AMF secretion is regulated, at least in part by HER2 signaling in breast carcinoma cells (Fig. 5A). AMF knockdown did not affect HER2 expression, whereas reduced p-HER2 levels along with suppression of AKT/ERK activation in T47D cells (Fig. 5B). In addition, the blocking of AMF function partially inhibited the phosphorylation of HER2, resulting in the reduction of p-AKT/p-ERK levels (Fig. 5C) and endogenously secreted AMF does not significantly affect the cleavage of HER2 in HER2-overexpressing cells (Fig. 5C).

Finally, to further convince that AMF–HER2 relationship is not only an in vitro phenomena, we conducted 2 independent immunohistochemistry studies using human breast cancer tumors and MCF10A DCIS.com xenograft, which is a mouse model of human comedo ductal carcinoma in situ (28). Compared with the surrounding stroma, both HER2 and AMF were found to be strongly expressed in the human breast carcinoma epithelium, and especially colocalized on the cell membranes of MCF10A DCIS.com xenograft (Fig. 5D).

The studies presented earlier show that exogenous AMF contributes to Herceptin/trastuzumab resistance of human breast carcinoma cells via generation of p95HER2 fragment. Because AMF interacts with HER2 and AMF-interacting HER2 is sensitive to HER2 shedding by MMPs, we hypothesize that the interaction between AMF and HER2 could structurally expose the MMPs enzymatic clip site at HER2 juxtamembrane region. The expression levels and activation of MMPs might be considered a critical limiting step of HER2 shedding due to partial recovery by AMF in trastuzumab-inhibited cell growth and motility (Fig. 4C and D). Despite the inhibition of MMPs, presence of AMF was sufficient for phosphorylation of HER2 and activation of PI3K/MAPK (Fig. 2C and D), suggesting that AMF can act as a HER2-binding partner enhancing its activation. Weather AMF-interacting HER2 underdoes heterodimerization with another member of HER2 family for PI3K/MAPK activation is yet to be determined.

Because both AKT and ERK activation occurs mostly in the cellular signaling of receptor tyrosine kinase (RTK), the mechanism in which gp78/AMFR knockdown showed the suppression of AMF-activated ERK but not AMF-induced AKT is poorly understood (Supplementary Fig. S1). Of note, gp78 signaling-suppress ERK activation and also acts as an E3 ligase in the cytoplasm (29).

Transgenic mouse models harboring rat full-length p185HER2 (Neu) recapitulated the initial events of HER2-induced mammary tumorigenesis. A long latency period for tumor initiation is required for the acquisition of activating mutations such as in-frame deletion or insertion at the juxtamembrane region of Neu transgene (30–32). As these mutations have not been found in human breast cancer, several studies have suggested epigenetic mechanisms for the expression of truncated p95HER2, for example, alternative translation initiation or an alternative spliced form of HER2 resembling Neu-derived somatic mutations (33–35). In this respect, p95HER has been found in subgroups of human patients with breast carcinoma, resulting in constitutively hyperactive dimers with intermolecular disulfide bonds (26, 36). Transgenic recapitulation studies showed that p95HER2 transgene-induced tumors lead to more aggressive and metastatic phenotype with relatively short latency period compared with full-length HER2-induced tumors (37). p95HER2 fragments are suggested to arise through mechanisms of proteolytic shedding by MMPs or translation of HER2 internal initiation codons (26, 38). In the present study, AMF-enhanced HER2 shedding suggests that AMF participates in HER2 oncogenic and aggressive progression through the epigenetic event of AMF secretion. As HER2 overexpression positively controls AMF secretion (Fig. 5A), the positive feedback loop of HER2 overexpression, AMF secretion and p95HER2 generation could be speculated in HER2-induced cancer progression. AMF secretion may also be controlled by other mechanisms, as the secretion level of AMF is higher in high motility cells (MDA-MB-231) compared with low motility cells (T47D), regardless of the similar expression levels of HER2 by both the cell lines (Supplementary Fig. S4).

Although the mechanisms of resistance to Herceptin/trastuzumab by breast tumors have been delineated at a preclinical level, it is still to be established in patients and remain a challenge for the clinicians. In vitro studies suggested Herceptin/trastuzumab could inhibit the shedding of p185HER2 (36, 39) and retrospective studies in patients with breast cancer revealed a high association between the predisposition of p95HER2 and clinical resistance to Herceptin/trastuzumab (8, 9). Previously, AMF was identified as a serum tumor marker in patients with breast cancer (18). It was also reported that Herceptin could effectively block both ligand-induced and constitutive expression of AMF associated with high HER2 overexpression, implying a role of the AMF pathway in the action of Herceptin (40). Here, we have documented that AMF treatment of breast carcinoma cells suppresses the effect of Herceptin/trastuzumab by overcoming both p-AKT/p-ERK levels and protecting cell growth through p95HER2 generation (Fig. 4). We suggest that high AMF-secreting HER2-positive cancer cells could initially and intrinsically have the advantage of resistance to Herceptin/trastuzumab. Therefore, we propose that AMF might be a novel therapeutic target in patients with HER2-overexpressing breast cancer and this target should be considered in combination with Heceptin/trastuzumab therapy. In summary, AMF might play a role in HER2-driven breast cancer progression and initial resistance of trastuzumab as depicted in Fig. 6.

No potential conflicts of interest were disclosed.

Conception and design: D.H. Kho, A. Raz

Development of methodology: D.H. Kho, L. Tait

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.H. Kho, V. Balan, L. Tait

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.H. Kho, V. Balan, L. Tait, A. Raz

Writing, review, and/or revision of the manuscript: D.H. Kho, P. Nangia-Makker, V. Balan, V. Hogan, Y. Wang, A. Raz

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): V. Hogan, Y. Wang, A. Raz

Study supervision: A. Raz

The authors thank Drs. Wei-Zen Wei and Arun Rishi at Wayne State University for the gift of trastuzumab, SkBr3, and BT474 cells, respectively. The authors also thank Dr. P.V.M. Shekhar for critical evaluation of the article.

This work was supported by NIH/National Cancer Institute R01 CA51714 (A. Raz).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.