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ErbB2 (HER2, neu) is a receptor tyrosine kinase overexpressed in about 25% of invasive breast carcinomas. Neutrophil gelatinase-associated lipocalin (NGAL) is a secreted glycoprotein expressed in a variety of cancers including breast carcinomas. NGAL can inhibit erythroid cell production leading to anemia. Anemia usually occurs in cancer patients and negatively impacts quality of life. However, current treatment for cancer-related anemia has potential complications. ErbB2, NGAL, and anemia have all been associated with increased metastasis and poor prognosis in breast cancer patients, although the relationship between ErbB2 and NGAL expression is not clear. Here, using breast cancer cell lines in vitro and transgenic mice carrying the activated c-neu oncogene driven by a mouse mammary tumor virus (MMTV-neu) in vivo, we demonstrate that ErbB2 overexpression leads to NGAL upregulation, which is dependent on nuclear factor kappa B (NF-κB) activity. MMTV-neu transgenic mice developed anemia after tumor onset, and anemia progression could be partially arrested by an NF-κB inhibitor and an ErbB2-targeted therapy. Taken together, upregulation of NGAL by ErbB2 through NF-κB activation is involved in cancer-related anemia, and ErbB2, NF-κB, NGAL pathway may serve as potential therapeutic targets for cancer-related anemia.
ErbB2 overexpression has been correlated with poor prognosis and increased incidence of metastasis in breast cancer patients (1). Anemia commonly occurs in breast cancer patients, and is also associated with poor prognosis, in addition to profound negative impact on patients’ quality of life (2). Current treatment for cancer-related anemia is blood transfusion or using erythropoiesis-stimulating agents; each has a potential risk of severe complications (3). Blood transfusions can result in infectious disease transmission and transfusion-related reactions (3), and recent phase III trials revealed erythropoiesis-stimulating agents increased the risk of developing venous thromboembolism and decreased survival rates in anemic cancer patients (4). Therefore, it is necessary to develop new treatment modalities for cancer patients with anemia.
Neutrophil gelatinase-associated lipocalin (NGAL; also known as LCN2 or lipocalin 2) is a 25kDa monomer capable of homo-dimerization and hetero-dimerization with neutrophil gelatinase (5). NGAL, identified as a component of neutrophil granules, inhibits bacterial growth by depleting their intracellular iron stores (6). NGAL is also a promising biomarker of kidney injury that predicts acute renal impairment (7). NGAL overexpression is found in a variety of cancers including breast carcinomas (8), and is a predictor of poor prognosis and increased risk of metastasis in breast cancer patients (8). Additionally, NGAL was shown to induce apoptosis of primary bone marrow cells including erythroid progenitor cells, and inhibit erythroid cell production leading to anemia (9-12). ErbB2, NGAL, and anemia have all been associated with poor prognosis in breast cancer patients. However, the association between ErbB2 and NGAL is debatable (8, 13). We postulated that ErbB2 overexpression may upregulate NGAL and play a role in cancer-related anemia. Indeed, we found that ErbB2 overexpression upregulates NGAL in a NF-κB-dependent manner. Additionally, MMTV-neu transgenic mice developed anemia after mammary tumor onset around 24 weeks mean time. Furthermore, NGAL expression and anemia progression could be partially inhibited by blocking the NF-κB pathway or by ErbB2-targeted therapy.
231BR.vec, 231BR.erbB2, MCF7.vec, and MCF7.erbB2 cells were described previously (14, 15). Antibodies used are NGAL(HYB 211-01; Antibodyshop), NGAL(M-145; Santa Cruz), Neu(C-18; Santa Cruz), β-actin (Sigma), phosphor-IκB-α (cell signaling), and IκB-α (cell signaling).
Immunobloting was performed as previously described (16).
The pGL3-NGAL-267 plasmid was obtained by PCR amplification of genomic DNA using forward primer 5′-CCACATACAGGGCAATCAGA-3′ and reverse primer 5′-GATTTCAGGGCCGAGGAAG-3′. This PCR fragment was ligated into pCR 2.1-TOPO II TA cloning vector (Invitrogen), then cloned into pGL3-basic (Promega) using the HindIII and XhoI restriction enzymes. The pGL3-NGAL-267(−267 to +69) plasmid contains 336bp from the region upstream of the translational start site on the LCN2 gene. All plasmids were sequenced across the multiple cloning sites of pGL3-basic to verify sequence integrity. Truncated deletion promoter plasmids, pGL3-NGAL-154(−154 to +69), and pGL3-NGAL-138(−138 to +69) were generated by PCR using pGL3-NGAL-267 as the template with the primers 5′-CTCTGTCTTGCCCAATCCTG-3′ and 5′-CCTGACCAGGTGCAGAAATC-3′ respecitively.
Collected after the indicated breast cancer cells were cultured for 48 h in serum-free DMEM/F12 to 80% to 90% confluency and concentrated using centrifugal filter units (Millipore).
Indicated cells were treated with trastuzumab 2μg/mL (Genentech Inc.) or human normal IgG (Jackson ImmunoResearch Laboratories, Inc.) for 6h and NF-κB inhibitor, Bay 11–7082 (Sigma), for 4 h. Conditioned media was collected 48h later and assayed as indicated.
Luciferase assays was performed as previously described (17).
Chromatin immunoprecipitation (CHIP) assay was performed as previously described (17). Antibodies used were IgG, histone H1, NF-κB p50 and p65 (all from Santa Cruz). Coprecipitated DNA (2 μL) was analyzed by PCR (30–35 cycles) using specific primers, 5′-AAGGCACAGAGGGAGTCGTTGTCC-3′ and 5′-AAGAGGAGGTGGCGAGTGAGAGGC-3′, to the NF-κB binding region of the NGAL promoter.
Immunohistochemistry was done as previously described (16).
MMTV-neu transgenic mice were obtained from Dr. William Muller, McGill University, Canada. MMTV-polyoma middle T antigen transgenic mice (PyMT) and FVB wild-type mice were obtained from Jackson Laboratory. Tail vein blood (200 μL) from mice was collected in microcontainers containing EDTA at the indicated time. Whole blood was processed for complete blood cell (CBC) counts by the Veterinary Laboratory Medicine Core Facility, Department of Veterinary Medicine and Surgery at The University of Texas M. D. Anderson Cancer Center. MMTV-neu mice were palpated for mammary tumors twice a week. Bay 11–7082 (5mg/kg) was dissolved in 0.5% methyl cellulose and injected intraperitoneally three times a week for 2 weeks (200 μL/mouse). The same amount of dimethyl sulfoxide dissolved in 0.5% methyl cellulose was used as the control for Bay 11–7082. The monoclonal antibody (MAb) 7.16.4 (2mg/kg) was injected intraperitoneally twice a week for 2 wk, and mouse IgG was used as controls. Protein extraction from mammary tumors was done as previously described (16).
Statistical differences were assessed with paired t test, unpaired t test, or one-way ANOVA as indicated. A p<0.05 was considered statistically significant.
To determine if overexpression of ErbB2 induces NGAL expression, we compared NGAL protein levels in conditioned media from MDA-MB-231BR and MCF7 human breast cancer cells stably overexpressing ErbB2 (231BR.erbB2 and MCF7.erbB2) or vector controls (231BR.vec and MCF7.vec). We found that ErbB2 high-expressing 231BR.erbB2 and MCF7.erbB2 cells secreted more NGAL than ErbB2 low-expressing vector control cells after serum starvation (Fig. 1A). To further investigate whether ErbB2 overexpression induces NGAL, we compared the NGAL expression in mammary tumors from MMTV-neu transgenic mice with that from MMTV-PyMT transgenic mice. Mammary tumors from MMTV-neu mice had higher NGAL expression than MMTV-PyMT mice (Fig. 1B), indicating ErbB2 overexpression induced NGAL expression.
To determine whether ErbB2 was required for NGAL upregulation, we treated 231BR.vec and 231BR.erbB2 cells with trastuzumab, an ErbB2 antibody, to inhibit ErbB2 signaling. Indeed, trastuzumab treatment reduced NGAL production along with ErbB2 downregulation in 231BR.erbB2 cells compared to the IgG control (Fig. 1C). Next, we tested if downregulation of ErbB2 in MMTV-neu mice could lead to decreased NGAL expression. When tumors became palpable, MMTV-neu mice were injected with either MAb 7.16.4 targeting the rat neu oncogene, or mouse IgG. Similar to trastuzumab treatment of 231BR.erbB2 cells, MAb 7.16.4 treatment of tumor-bearing MMTV-neu mice led to ErbB2 downregulation and reduced NGAL expression compared to IgG-treated controls (Fig. 1D). Together, these data indicate that ErbB2 overexpression induces NGAL upregulation.
To investigate the mechanism of ErbB2-mediated NGAL upregulation, we examined NGAL mRNA levels in 231BR.vec and 231BR.erbB2 cells by RT-PCR after serum starvation. The 231BR.erbB2 cells had increased NGAL mRNA levels compared to 231BR.vec cells (Fig 2A). To determined whether increased NGAL mRNA was due to transcriptional upregulation, we cloned the proximal region of NGAL promoter known to be involved in NGAL transcriptional regulation into a luciferase reporter (pGL3-NGAL-267) (Fig 2B). Relative luciferase activity was higher in 231BR.erbB2 cells transfected with pGL3-NGAL-267 than in 231BR.vec cells, indicating NGAL upregulation at transcriptional level (Fig 2C, left). To further identify the critical promoter region(s) for NGAL upregulation, we generated two NGAL promoter 5′ deletion constructs pGL3-NGAL-154 and pGL3-NGAL-138. 231BR.erbB2 cells failed to induce NGAL promoter-driven luciferase activity when NGAL promoter is deleted to −154 (Fig. 2C, right), indicating that the sequence between −267 and −154 is necessary for erbB2-mediated NGAL transcriptional upregulation. Among the sequences between −267 and −154, the sequences between −180 to−171 has a homology to the NF-κB binding-site consensus sequence 5′-GGG(A/G)NNT(C/T)CC-3′ (18). Indeed, CHIP assay showed that NF-κB subunits p50 and p65 bind to the NGAL promoter in 231BR.erbB2 cells after serum starvation (Fig 2D).
To further determine the contribution of NF-κB in erbB2-mediated NGAL upregulation, we treated 231BR.erbB2 cells with Bay 11–7082, which inhibits IκBα degradation and blocks NF-κB translocation from the cytoplasm to the nucleus. Bay 11–7082 treatment decreased NGAL levels in the conditioned media from 231BR.erbB2 cells by inhibiting NF-κB binding to the NGAL promoter (Figs. 3A&B). Furthermore, treatment of tumor-bearing MMTV-neu mice with Bay 11–7082 reduced phospho-IκBα, NF-κB nuclear localization, and NGAL expression in mammary tumors relative to control-treated mice (Fig. 3C&D). These data indicate that NF-κB signaling is important for ErbB2-mediated NGAL upregulation.
To test if anemia occurs in tumor-bearing MMTV-neu mice, we collected blood from the tail veins and analyzed the CBC counts before mammary tumor development at 16 wk of age and again when mammary tumors were palpable at 24wk. As controls, we also counted CBC from the wild-type mice at 24 wk of age. After tumor onset, MMTV-neu mice had significantly (p< 0.01) lower hemoglobin levels than wild-type mice and MMTV-neu mice before their tumors appeared (Fig. 4A). To investigate whether blocking the NF-κB pathway can impede anemia progression in tumor-bearing MMTV-neu mice and MMTV-PyMT mice, we treated mice intraperitoneally with Bay 11–7082 or placebo. CBC counts before and after treatment demonstrated that anemia progression could be partially reversed by Bay 11–7082 treatment in MMTV-neu mice, but not in MMTV-PyMT mice (Fig. 4B). Additionally, the ErbB2-targeted therapy MAb 7.16.4 also partially arrested the anemia progression in tumor-bearing MMTV-neu mice (Fig. 4C).
In this study, we found that ErbB2 induces NGAL expression through NF-κB activation that contributes to anemia in mice with ErbB2/neu-overexpressing tumor; whereas a NF-κB inhibitor and erbB2-targeted therapy can decrease NGAL expression in ErbB2/neu-overexpressing mammary tumor and partially reverse anemia progression. Previous studies reported that NGAL can induce erythroid progenitor cell apoptosis, inhibit red blood cell production, and lead to anemia (10-12). NF-κB is also responsible for impaired erythropoietin synthesis which contributes to the development of cancer-related anemia (19). Our findings are consistent with previous reports, thus we propose that both NGAL and NF-κB may play critical roles in cancer-related anemia, especially, in ErbB2/neu-overexpressing tumor-related anemia. Anemia negatively influences cancer patients’ overall quality of life and worsens their prognosis. Unfortunately, current treatment for cancer-related anemia has limitations. Since NF-κB inhibitor is currently under clinical trials (20) and ErbB2-targeted therapy (trastuzumab) is routinely used for patients with ErbB2-overexpressing breast cancers (1), our data provide a rational base for using these targeted therapies as potential new treatments for cancer-related anemia, which could impact on breast cancer patient care. Meanwhile, further studies are necessary to elucidate the underlying mechanism of NGAL and NF-κB in cancer-related anemia and identify better therapeutic targets as the NF-κB inhibitor or ErbB2-targeted therapy had partial efficacy in treating cancer-related anemia in MMTV-neu mice. In conclusion, our data provided evidence that NF-κB plays a pivotal role in ErbB2-mediated NGAL upregulation and cancer-related anemia. This pathway may serve as promising therapeutic targets itself and provide insight for further identification of other targets.
Financial support: This work is supported by NIH grants P30-CA 16672 (MDACC), RO1-CA109570, RO1-CA112567, PO1-CA099031 project 4, P50 CA116199, project 4, DOD Center of Excellence grant subproject W81XWH-06-2-0033, DOD Synergistic Award W81XWH-08-1-0712 (DY), and Susan G. Komen Breast Cancer Foundation Promise Grant KG091020 (D. Yu). D. Yu is the Nylene Eckles Distinguished Professor in Breast Cancer Research at M. D. Anderson Cancer Center.
S.-H. L. is partially sponsored by Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan.
Conflicts of interest: None