Molecular Targeting of RRM2, NF-κB, and Mutant TP53 for the Treatment of Triple-Negative Breast Cancer

Falling under the basal-like classification, triple-negative breast cancer (TNBC) is defined as lacking three specific receptor types: estrogen receptor (ER), progesterone receptor, and HER2. Although the HER2⁺ and HR⁺ cancer types may be effectively treated with targeted anti-HER2 or hormonal treatments, the basal-like and TNBC groups are much more challenging to treat due to their general lack of essential receptors for drug targeting (1).

Although definitive targets for TNBC remain elusive, there are certain immunotherapies or targeted therapies that may improve patient survival. PARP, programmed cell death protein 1 (PD-1) and its ligand (PD-L1), receptor tyrosine kinase targets (such as VEGF, EGFR, FGF/FGFR), and MEK and AKT pathways are among the most common current targets for the treatment of TNBC (2–6). However, some of these treatment modalities encompass major drawbacks and toxicities with some patients failing to respond to treatment, whereas others have a response that is short lived with resistant growth subsequently occurring, and others seem promising, but trials end with no available published data. In addition, the cost of the therapeutics for these patients is particularly high despite only minor increases in overall patient survival rates (7–10). In this article, we focus on ameliorating the toxicity of current chemotherapy as well as enhancing the standard of care for this difficult-to-treat population of patients with TNBC.

Specific activity of ribonucleotide reductase (RR), which is an enzyme that catalyzes the rate-limiting step in DNA synthesis converting ribonucleotides into deoxyribonucleotides, has been previously correlated with tumor growth rates (11). RR is present as a heterodimeric tetramer consisting of two subunits, RRM1 and either RRM2 or p53R2 (12). Overexpression of RRM2 has been linked to higher proliferation and invasiveness of malignant cells (7, 13). Previously, we have shown that RRM2 is upregulated in ER-negative as well as tamoxifen-resistant ER-positive breast cancers (14, 15). Others have also shown the upregulation of RRM2 in breast cancer, as well as suggesting it to be a possible prognostic indicator (8, 16–20).

The tumor protein p53 (TP53 or p53) prevents tumorigenesis by maintaining genome integrity and preventing the proliferation of cells with a damaged genome (21). In response to cellular stresses such as DNA damage, oncogene expression, or ribosome dysfunctions, p53 becomes posttranslationally modified, stabilized, and activated. Once activated, p53 triggers transcription of an important number of direct target genes mainly implicated in cell-cycle arrest (such as CDKN1A/p21), DNA repair, apoptosis, and senescence as well as to enhance metabolic changes and antioxidant responses (22). However, mutated p53 may actually contribute to tumor progression by a loss of tumor suppression as well as a gain of oncogenic activity (23). Somatic mutations in the TP53 gene occur in almost every type of cancer at rates up to 50% and are more frequent in advanced stage or in cancer subtypes with aggressive behavior (23). In particular, the TP53 gene was found to be mutated in approximately 80% of TNBC, and high levels of p53 in TNBC have been associated with poor prognosis (24).

The synthetic antioxidant and potent RR inhibitor didox (3,4-dihydroxybenzohydroxamic acid) was originally developed as an antineoplastic and antiproliferative agent to improve upon the activities of hydroxyurea (25). Didox (DDX) is a bifunctional compound that possesses both iron-chelating and free-radical scavenging functions and has proven enhanced efficacy when used in combination with DNA-targeting agents (9, 25, 26). Specifically, the use of DDX has been shown to display synergism when used in combination with the DNA agent doxorubicin (DOXO) by inactivating nuclear transcription factor NF-κB, increasing intercellular DOXO concentrations, and facilitating apoptosis (27). In addition, DDX displays a favorable side effect profile when used at therapeutic concentrations (28).

In this study, we show that through the inhibition of RRM2, mutant p53, and the suppression of the NF-κB pathway, DDX works synergistically with DOXO in order to halt malignant TNBC cell division, both in vitro and in vivo. We also show that this combination therapy has the potential to reduce anthracycline-associated cardiomyopathy as indicated by in vivo increased heart mass and left ventricle diameter. Interestingly in breast cancer cells with mutant p53, we observed that DDX therapy is effective in targeting or downregulating mutant p53. Yet, it does not alter total wild-type (WT) p53 in cells with functioning p53. Our data present a novel and promising approach for the treatment of TNBC that merits further clinical evaluation in human models.

DDX was synthesized and kindly provided by Dr. Howard L. Elford, Molecules for Health (25). DOXO hydrochloride for cell experiments was obtained through VWR. DOXO hydrochloride (2 mg/mL) for in vivo experiments was obtained from APP Pharmaceuticals, LLC. All of the compounds were dissolved in 0.9% sterile saline solution, filtered through a 0.45-mm syringe filter, and stored at 40°C in the dark for a maximum of 1 week.

MDA-MB-468, MDA-MB-231, BT20, MCF7, and ZR751 cells were routinely purchased from the ATCC every 6 months. Cells were maintained in advanced DMEM/F12 (Fisher) supplemented with 5% FBS, 1% l-glutamine, 1% streptomycin, and penicillin. Cells were treated with 600 μmol/L DDX, 100 nmol/L DOXO, or a combination of 600 μmol/L DDX and 100 nmol/L DOXO in phenol-red–free, serum-free, DMEM/F12 for 24 hours and compared against a control sample consisting of no treatment (NT). In the dose-dependent study, DDX concentrations between 30 and 900 μmol/L were used.

CellTiter-96 Aqueous One Solution (Promega) was used according to the manufacturer's protocol to determine cell viability and proliferation. One thousand cells in DMEM/F12, phenol-red–free medium supplemented with 2% charcoal-striped serum (CSS, Fisher) were seeded into each well of a 96-well plate. After 24 hours, cells were treated as stated above and treatment media replenished on days 2, 4, and 6. Aqueous One Solution was added to each well, incubated for 1 hour, and analyzed at 490 nm.

IC₅₀ values for DOXO and DDX were determined for MDA-MB-468 and MDA-MB-231 cell lines. Several fixed dose ratios of DOXO:DDX were then calculated and used to determine the combination indices (CI) of the drug combinations using the method developed by Chou and Talalay. Synergy, additivity, and antagonism are indicated by CI values of <1, 1, and >1, respectively (29).

After transfecting MDA-MB-468 cells with NF-κB vector (QIAGEN), cells were treated with varying concentrations of DDX. NF-κB transcriptional activity was measured through the use of the Dual Luciferase Assay (Promega) system and according to the manufacturer's protocol. Briefly, 10,000 cells in DMEM/F12, phenol-red–free medium supplemented with 2% CSS were seeded into each well of a 96-well plate and transfected 24 hours thereafter. Treatments were added 24 hours after transfection, and luciferase activity was measured after 18 hours of treatment time.

Cells and tumor samples were disrupted in RIPA buffer (Sigma), and lysates were clarified by centrifugation for 15 minutes at 15,000 × g. After the protein concentration was determined, an equal amount of total protein for each sample was loaded for Western blotting. RRM2 (Sigma), γ-H2AX, Bcl_XL, Bcl2, caspase 3, PARP, cleaved-PARP, p21, Pp53S392, Pp53 S15, p53, IKK-α, IKK-β, p-IkBα, IkBα, p52, P-p65, p65, p100, p105, RelB, C-Rel, AKT, P-AKT, GAPDH (Cell Signaling Technology), apoptosis sampler kit (Cell Signaling Technology), NF-κB family members sampler kit (Cell Signaling Technology), NF-κB pathway sampler kit (Cell Signaling Technology), and pγ-H2AX (Millipore) were detected and visualized once incubated with secondary donkey anti-rabbit or goat anti-mouse IgG antibody conjugated with IRDye 800CW or 680RD (LI-COR Biosciences) for 2 hours at room temperature. Membranes were subsequently washed and visualized using a LI-COR Odyssey Imaging System, Image studio.

All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of the Pacific. A total of three experiments were performed: MDA-MB-468 was injected subcutaneously into the flank of nude female mice (27–33 tumors/group/study). As indicated, mice received DOXO (intravenous, 2–10 mg/kg weekly), DDX (intraperitoneal, 425 mg/kg daily), or a combination of both. For the DDX injections, solutions were made daily and injected fresh. Tumor volumes were measured using the ellipsoid formula of [4/3π(r1)²(r2)], where r1 < r2. Body weights were taken weekly with no changes in body weight observed in the DDX group. Tumors were collected and weighed at study termination, snap-frozen in liquid nitrogen, and stored at −80°C for Western analysis. Whole hearts were also harvested at the end of the study, their mass measured, and either snap-frozen for Western analysis or formalin-fixed for IHC staining. Blood samples were also collected postmortem and centrifuged (14,000 rpm × 10 minutes, 4°C) in order to obtain individual serum samples.

Serum samples were assayed for troponin T in duplicate using an ELISA (Enzyme–Test cTnT). Troponin T assay was from LSBIO and performed by the National Mouse Metabolic Phenotyping Center at the University of California, Davis. Data can be found at https://www.mmpc.org/shared/phenotype/showAssay.aspx?id=1004.

The expression of RRM2 in patients with TNBC was examined using the GEO2R tool from three independent microarray datasets submitted to the gene expression omnibus. The expression of RRM2 was significantly elevated in patients with TNBC for all three independent datasets (Supplementary Fig. S1A). These results were also observed when staining TNBC patient tissue samples for RRM2 in which we observed that RRM2 levels increase in tumors of patients with TNBC with stage progression (Supplementary Fig. S2A–S2D). In addition, a statistically significant difference in relapse-free survival (RFS; Supplementary Fig. S1B) and overall survival (OS; Supplementary Fig. S1C) was observed between TNBC groups with high versus low expression of RRM2. Interestingly, in the presence of mutant p53, levels of RRM2 become less predictive of RFS (Supplementary Fig. S3B) and OS (Supplementary Fig. S3D), when compared with patients with WT p53 (Supplementary Fig. S3A and S3C).

Previously, the RR inhibitor DDX has been shown to display synergism when used in combination with the DNA-damaging agent DOXO (27). Here, we tested the effect of adding DDX to DOXO therapy on TNBC cell growth. Cell proliferation over 7 days was examined after DOXO alone or in combination with DDX treatment as compared with untreated MDA-MB-231 and MDA-MB-468 TNBC cells. The combination of 26 μmol/L DDX and 50 nmol/L DOXO was found to decrease cellular proliferation as compared with the same dose of the single-agent DOXO for both cell lines (Fig. 1A and B). Next, these cells were treated with varying concentrations of DOXO alone or combined with 26 μmol/L DDX, and IC₅₀s were determined (Fig. 1C and D). For the MDA-MB-231 cell line, drug combinations of DDX and DOXO of less than 20.5 μmol/L and 50.5 nmol/L were found to be synergistic, with CI values of 0.87 for 20.5 μmol/L DDX/50.5 nmol/L DOXO and 0.65 for 10.3 μmol/L DDX/25.3 nmol/L DOXO. All tested DOXO/DDX concentrations exhibited synergism in the MDA-MB-468 cell line, with CI values ranging from 0.53 for 212 μmol/L DDX/748 nmol/L DOXO to 0.37 for the 13.3 μmol/L DDX/46.8 nmol/L DOXO combination (Fig. 1C–F). Proliferation assays showed that DDX in combination with DOXO inhibited TNBC cell growth in a time- and dose-dependent manner.

Based on the data from the proliferation and synergism assays, we chose the MDA-MB-468 TNBC cell line for the subsequent studies described here. Because DDX has been shown to display synergism when used in combination with DOXO by downregulating the activation of the nuclear transcription factor NF-κB, we wanted to determine the concentrations of DDX that would decrease NF-κB–related proteins and activity in TNBC cells. Therefore, DDX concentrations ranging from 30 to 900 μmol/L were used to treat MDA-MB-468 cells for 24 hours. DDX reduces the expression of RRM2 and the NF-κB proteins p52 and p100 as well as the related proteins IKK-β and Rel B. Interestingly, DDX also reduced the total p53 and S15 phosphorylated p53. Caspase 3 was also downregulated as DDX exposure increased (Fig. 2A). DDX concentrations ranging from 30 μmol/L to 30 mmol/L were used to assess NF-κB activity in the MDA-MB-468 cell line. Although concentrations of 30 to 150 μmol/L displayed no significant change, all higher concentrations displayed significant downregulation in NF-κB activity (Fig. 2B).

Next, MDA-MB-468 cells were exposed to varying treatment times of 600 μmol/L DDX (15 minutes–24 hours) and compared with control (24 hours of NT). Treatment with DDX reduced the expression of p53 beginning as early as 6 hours and more significantly at the 12- and 24-hour time points, compared with vehicle control. Total IκBα remains unchanged as didox-timed exposure was increased. Total H2AX expression progressively increased through the 24-hour DDX time period, and phosphorylated H2AX only expressed a band at 24 hours of DDX treatment (Fig. 2C).

To evaluate the effect of DDX and DOXO combination therapy on the IKK, NF-κB, growth receptor signaling, and apoptosis pathways, MDA-MB-468 cells were treated with vehicle (NT), 600 μmol/L DDX, 100 nmol/L DOXO, and 600 μmol/L DDX + 100 nmol/L DOXO for 24 hours. IKK signaling proteins were all downregulated with DDX treatment in the DDX alone as well as DDX+DOXO groups (Fig. 3).

Significant downregulation in the NF-κB p52, p65, p100, p105, RelB, and C-Rel proteins was observed in the DDX and DDX+DOXO groups. Similarly, both total AKT and p53 were downregulated in the DDX and DDX+DOXO groups compared with NT and DOXO alone. Phosphorylation of AKT and total p21 expression remained consistent across all groups, whereas phosphorylated p53 (both S392 and S15) were significantly downregulated in both the DDX and DDX+DOXO groups (Fig. 3).

Nude mice were injected with TNBC MDA-MB-468 cells in order to determine the effects of the DDX/DOXO combination in vivo. Tumors were allowed to reach an average size of 64 mm³ before treatments began (day 0). In the vehicle (NT) treatment group, mice tumor volumes reached an average tumor volume of 217 mm³ at the 31-treatment day mark, whereas DOXO-treated mice saw an average tumor volume of only 68 mm³. Animals treated with the DDX/DOXO combination were significantly smaller tumors that had an average tumor volume of 16 mm³ (Fig. 4A). All animals survived until takedown at posttreatment day 31. Tumors were collected, weighed, and snap-frozen at study end. Tumors of mice that had been treated with DDX+DOXO displayed significantly smaller masses than their DOXO-treated counterparts (P < 0.05, Fig. 4B).

Tumor xenografts were analyzed for protein expression of key signaling proteins in the IKK, NF-κB, and apoptosis pathways. Tumors displayed reduced IKK-β as well as phosphorylated and total IκBα in the DDX+DOXO tumors. Reduced expression of the NF-κB proteins p50, total p65, p100, p105, RelB, and C-Rel was observed in the DDX+DOXO group when compared with the NT-, DDX-, and DOXO-treated animals. RRM2 levels in conjunction with phosphorylated and total p53 were also significantly reduced in the DDX+DOXO-treated tumors (Fig. 4C).

To evaluate animals for DOXO-induced cardiac damage, serum from NT-, DDX-, and DDX+DOXO-treated animals was collected and analyzed for troponin T. Increased levels of troponin T were observed in the DOXO group as compared with that of NT- and DDX+DOXO-treated animals. There were no statistically significant differences in troponin T levels between the NT and DDX+DOXO groups (P > 0.05, Fig. 4D).

At the end of the study, the hearts of all mice were collected, weighed, and sectioned. DOXO-treated hearts displayed a significant increase in heart mass when compared with all other treatment groups (P < 0.05, Fig. 4E). Left ventricular thickness also appeared enlarged in the DOXO group compared with other treatment groups (Fig. 4F). There were no significant differences between the NT- and DDX+DOXO-treated heart masses or left ventricle diameters (P > 0.05).

Examination of signaling pathways revealed that total and phosphorylated p53 protein levels are significantly decreased in MDA-MB-468 cells after DDX treatment (Figs. 2A and C, 3, and 4C). To determine if this is an isolated cell type effect, a panel of breast cancer cell lines with WT p53 [MCF7, A3B5 (MCF7-overexpressing AKT), and ZR-75-1] and mutant p53 (BT20, MDA-MB-231, and MDA-MB-468) were analyzed for expression level of total and phosphorylated p53. DDX treatment resulted in downregulation of total p53 (as well as total mutant p53) in all three TNBC cell lines with mutant p53 (Fig. 5, bottom). The effect of DDX on decreasing phosphorylated p53 was more significant in MDA-MB-468 and BT20 cells as compared with MDA-MB-231 cells. In contrast, in breast cancer cell lines with WT p53, total p53 remained unchanged after DDX treatment. Yet, phosphorylated p53 was reduced with DDX treatment in breast cancer cell lines with WT p53 (Fig. 5, top).

Effective targets for this particularly virulent TNBC subtype are largely unknown. Here, we have demonstrated that coupling an RRM2 inhibitor with traditional anthracycline therapy is effective in the inhibition of TNBC tumors. Several studies have linked RRM2 overexpression in breast cancer to increased cell proliferation and invasiveness as well as conferring chemoresistance (8, 16, 20). We have previously observed that RRM2 is upregulated in TNBC subtypes when compared with ER-positive breast cancers, thus potentially providing a rationale for the targeting of RR. Here, we report that tumors of patients with TNBC exhibit higher RRM2 as compared with normal tissues (Supplementary Figs. S1A and S2A–S2D). Moreover, increased RRM2 expression in TNBC was observed to correlate with poorer outcomes (Supplementary Fig. S1B and S1C). When analyzing the TNBC microarray datasets for RRM2 expression levels, it appears that higher levels of RRM2 in WT p53 TNBC are predictive of lower RFS and OS (Supplementary Fig. S3A and S3C). However, no differences in RFS and OS were observed between high and low levels of RRM2 in the presence of mutant p53, with both RRM2 expression groups exhibiting lower probability curves (Supplementary Fig. S3B and S3D). It may be noted that the precise levels of RRM2 in these TNBC samples are unknown, and given our observations, we may predict that in mutant p53 TNBC, RRM2 expression levels already are high and exceed that of normal tissue.

NF-κB and associated NF-κB genes have been purported to be key regulators in TNBC (30–32). Responsible for cellular proliferation, survival, and apoptosis, NF-κB is activated by IκB kinases and tightly conserved through transcriptional regulation (33, 34). NF-κB activation has been established as a poor prognostic predictor for patients with TNBC treated with adjuvant anthracycline chemotherapy in addition to playing a role in DOXO chemoresistance (30–32). Specifically, DOXO was shown to induce NF-кB–dependent gene expression of migration, cell adhesion, and metastasis-related NF-кB target genes (31). We have found that the RR inhibitor DDX has the potential to alter the expression of several NF-κB proteins [NF-κB1 (p105/p50) and NF-κB2 (p100/p52)] that aid malignant cell survival and proliferation. Significantly decreased expression of RelB, NF-κB1 (p105), NF-κB2 (p100), and C-Rel was also present in the DDX + DOXO combination therapy tumors. Both IKK-β and total IkBα were similarly repressed in the combination therapy group (Fig. 3). We have also observed that at levels of 300 μmol/L and greater, DDX monotherapy attenuates NF-κB activity in vitro (Fig. 2B). This supports the role of DDX as not only an iron chelator and free radical scavenger that disrupts RRM2 activity, but also as an inhibitor of NF-κB activation.

WT p53 is widely acknowledged as the “guardian of the genome” and is responsible for a variety of tumor-suppressive effects, including DNA repair, cell-cycle arrest, and apoptosis (33–35). However, p53 is often found in a mutated form in a variety of breast cancers, including 80% of TNBC, and is linked to poor prognosis in these patients (36–39). In one study, mutated p53 mediated the DOXO induction of NF-кB–regulated gene transcription in TNBC (31). In addition, it has been previously noted that RRM2 levels are increased in the presence of mutant p53 (40). Therefore, the elimination of mutant p53 is of particular therapeutic interest in TNBC. We have found that through the administration of DDX, the mutant form of p53 is significantly reduced in vivo with MDA-MB-468 xenografts (Fig. 4C). Similarly, this reduction is observed in two additional mutant p53 cell lines, BT20 and MDA-MB-231, when treated with DDX in vitro (Fig. 5). Interestingly, this effect is observed in a dose- and time-dependent manner in vitro in MDA-MB-468 cells (Fig. 2A and C). Total and phosphorylated mutant p53 displayed decreased levels of expression in the combination therapy group in vivo, indicating further suppression of the prosurvival pathway known to be present in MDA-MB-468 cells (Fig. 4A).

We hypothesize that MDA-MB-468 cells rely heavily on the dysregulation/activation of the PI3K pathway due to their mutated form of PTEN and p53, in order to further cellular proliferation and oncogenesis. As given in Fig. 6A, PTEN and p53 cooperate to control cellular proliferation in normal breast tissue. However, in the presence of PTEN and p53 mutations (as in the MDA-MB-468 cells; Fig. 6B), levels of AKT and RRM2 are increased, promoting cellular proliferation. Because DDX is able to inhibit both mutant p53 and RRM2, decreased cellular proliferation in the presence of DOXO was observed in the MDA-MB-468 cell line.

In the two TNBC cell types, MDA-MB-231 and MDA-MB-468, analyzed in vitro, we have shown the combination of DDX and DOXO results in a synergistic inhibition of cellular proliferation (Fig. 1A–F). We also observed that the addition of daily DDX to DOXO therapy was superior to DOXO monotherapy in the suppression of MDA-MB-468 tumor growth in vivo (Fig. 4A). End tumor masses in the DDX + DOXO group were statistically smaller as compared with DOXO monotherapy (Fig. 4B, P < 0.05). In a previous phase II clinical study, DDX alone was administered as an infusion once every 3 weeks to patients with advanced breast cancer, with no signs of efficacy (41). Because DDX has a half-life of less than 45 minutes, it is not surprising that the dosing interval used in the clinical study resulted in a lack of a response, as we observed favorable efficacy outcomes when administering DDX daily in xenograft models.

In addition to its chemotherapeutic synergism potential, DDX is well known for its favorable safety profile (9). Here, we have demonstrated that the addition of DDX ameliorates the detrimental off-target effects of DOXO toxic metabolites on the myocardium. Presumably, DOXO injures the heart by generating damaging free radicals through iron-catalyzed redox cycling (42). We observed that overall heart mass in the DOXO monotherapy group was statistically increased when compared with the NT and DDX monotherapy groups, whereas the addition of DDX to DOXO was sufficient to protect against this hypertrophy (Fig. 4E). Troponin T levels, which are a known indicator of cardiac damage, were elevated in the DOXO monotherapy group when compared with the NT and DDX + DOXO combination group (Fig. 4D). Total heart and left ventricle cross-sections displayed enlargement with DOXO monotherapy as compared with combination therapy (Fig. 4F). The observed cardioprotective benefits of DDX are likely due to its iron-chelating characteristic that may protect against iron perturbations likely due to the DOXO alcohol metabolite doxorubicinol as well as its free radical scavenging characteristic that may protect against the oxidant activity likely due to DOXO deoxyaglycone and DOXol hydroxyaglycone (43). Specifically, DDX was shown to offer cardioprotection against DOXO-mediated injury through its ability to scavenge free radicals (44).

In summary, our findings regarding therapeutically targeting RRM2, NF-κB, and mutant p53 in TNBC complement the current breast cancer landscape. We have found evidence that increased levels of RRM2 and, as a consequence, greater NF-κB activation may be a hallmark of TNBC tumors, giving rise to their aggressive and difficult-to-treat nature. Reduced RRM2 and NF-κB pathway protein expression, as well as the overturn of the prosurvival mutant p53 through the use of DDX, results in reduced tumor size. We have observed that the addition of DDX also reduces the cardiotoxicity associated with anthracycline use, as evidenced by reduced heart mass and troponin T levels. Ultimately, our data add to the rationale that inhibitors of RRM2, NF-κB, and mutant p53 may be used to supplement traditional chemotherapies and offer improved efficacy and reduced toxicity.

H.L. Elford reports other from Molecules for Health outside the submitted work; in addition, H.L. Elford had a patent for Molecules for Health issued to Molecules for Health and is a major shareholder and employee of Molecules for Health. No potential conflicts of interest were disclosed by the other authors.

E.A. Wilson: Formal analysis, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. N. Sultana: Formal analysis, validation, investigation, visualization, methodology, writing–review and editing. K.N. Shah: Software, formal analysis, validation, writing–review and editing. H.L. Elford: Conceptualization, writing–review and editing. J.S. Faridi: Conceptualization, writing–review and editing.

This work was supported, in part, by a Graduate Student Research Grant (K.N. Shah), a SAAG intramural fellowship (J.S. Faridi), and the Thomas J. Long School of Pharmacy, University of the Pacific.

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