Functional Effects of GRM1 Suppression in Human Melanoma Cells

Malignant melanoma, the most deadly form of skin cancer, which is also the fifth most common cancer in men and the sixth most common cancer in women, poses a substantial clinical burden with more than 76,000 Americans estimated to be diagnosed and 9,000 succumbing to the disease in 2012 (1). Using a transgenic mouse model (2), our group identified that the ectopic expression of metabotropic glutamate receptor 1 (Grm1), in melanocytes was sufficient to induce spontaneous melanoma development (3). Grm1 is a G-protein–coupled receptor (GPCR) whose expression is normally localized to the central and peripheral nervous system and has functional implications on learning and memory formation (4). Analysis of human melanoma cell lines and biopsy samples has detected GRM1 expression in 80% of cell lines and 65% of primary to metastatic biopsy samples but not in normal melanocytes or benign nevi (5, 6). In addition, we showed that stable mouse melanocytic clones with exogenously transfected Grm1 exhibited transformed phenotypes in vitro and were tumorigenic in vivo (7). Furthermore, a conditional Grm1 transgenic mouse model confirmed the requirement for continuous expression of Grm1 in murine melanocytes for initial tumor formation and progression in vivo (8).

The metabotropic glutamate receptor family encompasses 8 receptors, which are divided into 3 groups according to agonist pharmacology, sequence homology, and transduction mechanisms via coupling to second messenger systems (4, 9–11). Group I GRM1 and GRM5 receptors are coupled to the activation of phospholipase C through G_q proteins; group II GRM2 and GRM3 receptors and group III GRM4, GRM6, GRM7, and GRM8 receptors are negatively coupled to adenylyl cyclase through G_i/0 proteins in heterologous expression systems (4, 9–11). In addition to GRM1, 2 other GRMs have been shown to have roles in melanoma development. It was recently shown that overexpression of Grm5 in mouse melanocytes can induce melanoma development in a transgenic mouse model (12). This overexpression of Grm5 was found to result in the activation of the mitogen-activated protein kinase (MAPK) pathway (12). Another group conducted a large-scale mutational analysis of GPCRs and identified mutations in GRM3 in approximately 17% of melanoma tumor samples (13). Some of these mutations were found to confer a growth advantage for the tumors through the action of MEK1/2, a kinase in the MAPK signaling pathway. In addition, GRM3 mutant melanoma cells were found to be more responsive to MEK inhibition with AZD-6244 than wild-type cells, especially when they also harbored BRAF^V600E mutations (13).

Taken together, these reports suggest that glutamate receptors and glutamatergic signaling may play greater roles than previously thought in melanoma biology. Given that the large percentages of human melanomas examined showed GRM1 expression, we were interested to know if suppression of GRM1 expression may modulate the growth of human melanoma cells in vitro and stimulation of downstream signaling cascades as well as tumorigenic potentials in vivo. Here, we showed that siRNA targeted to GRM1 suppressed expression of the receptor and led to decreased levels of activated mitogenic MAPK as well as anti-apoptotic PI3/AKT pathways resulting in reduced cell proliferation and increased apoptosis in vitro and in vivo.

Antibodies against GRM1 (Novus Biologics); phospho-AKT (S473), total-AKT, phospho-ERK1/2, total-ERK1/2, PARP, caspase-3, and cleaved caspase-3 (Cell Signaling); phospho-AKT 2 (S474; Genscript); α-tubulin (Sigma); anti-VgRXR-15C3 and 9B9 antibodies were a gift from the Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, IA); Zeocin, G418, blasticidin, hygromycin, Lipofectamine 2000, Plus Reagent, ViraPower Lentiviral Packaging mix (Invitrogen); doxycycline (Sigma); Ponasterone A (Enzo Life Sciences); sucrose (Fisher Scientific); DOTAP (Roche Applied Science); polybrene (Chemicon).

UACC903, 1205Lu, and C8161 were provided by Drs. Jeffery Trent (The Translational Genomics Research Center, Phoenix, AZ), Meenhard Herlyn (Wistar Institute, Philadelphia, PA), and Mary Hendrix (Children's Memorial Research Center, Chicago, IL). The cells were maintained in RPMI-1640 plus 10% FBS. HEK293T cells and H1299 cells were provided by Drs. Renping Zhou and C. S. Yang (Rutgers State University, New Brunswick, NJ) and were maintained in Dulbecco's Modified Eagle's Medium plus 10% FBS.

Tet receptor (TetR) cloned in lentiviral vector PLenti6L was a generous gift provided by Dr. Andrew Aplin (Kimmel Cancer Center, Philadelphia, PA). Lentivirus production was done using the ViraPower Packaging Mix according to the manufacturer's instruction.

C8161 cells were transfected with 4 μg of pVgRXR plasmid DNA (a gift from Dr. Danny Rangasamy, The Australia National University, Canberra, Australia) with Lipofectamine 2000 reagent as per the manufacturer's protocol. Stable clones expressing pVgRXR receptor were selected using 5 μg/mL of zeocin in RPMI plus 10% FBS. Receptor expression was confirmed by Western blotting. C8161 pVgRXR cells were transfected with 4 μg inducible siGRM1 plasmid DNA with the siRNA sequence 5′-GATGTACATCATTATTGCC-3′ (7), cloned into the pIND vector containing 5 repeats of ecdysone response elements (14) using Lipofectamine 2000. Stable C8161 pVgRXR siGRM1 clones were generated by selection with 5 μg/mL zeocin and 300 μg/mL G418. Induction of siGRM1 was achieved by treating the cells with 1 to 10 μmol/L of ponasterone A (PonA) for 7 days as described (15, 16).

UACC903 cells were transduced overnight with TetR lentivirus particles and 7.5 μg/mL polybrene. Stable UACC903 TetR cells were selected with 1 μg/mL blasticidin. Stable 1205 Lu TetR cells were a gift from Dr Andrew Aplin and were maintained in RPMI plus 10% FBS and 5 μg/mL blasticidin. UACC903 TetR and 1205 Lu TetR cells were transfected using DOTAP reagent with 4 μg of siGRM1 plasmid DNA with the sequence 5′ATGTACATCATTATTGCC3′ cloned into the pRNATin-H1.2/Hygro vector as described (7). Stable UACC903 TetR siGRM1 clones were generated by selection with 1 μg/mL blasticidin and 10 μg/mL hygromycin, whereas 1205 Lu TetR siGRM1 clones were selected with 5 μg/mL blasticidin and 100 μg/mL hygromycin. Induction of siGRM1 was carried out by treating the cells with 10 ng/mL of doxycycline for 7 days.

Protein lysates were extracted from C8161 pVgRXR, C8161 pVgRXR-siGRM1 A10, or C8161 pVgRXR-siGRM1 A13 treated with plain media, vehicle control (dimethyl sulfoxide; DMSO), or 10 μmol/L PonA as described previously (6) and assayed for the expression of GRM1, phosphorylated and total ERK1/2, tubulin, caspase-3, and cleaved caspase-3 with the corresponding antibodies. Similarly, protein extracts from UACC903 TetR, UACC903 TetR siGRM1-8, UACC903 TetR siGRM1-11, 1205 Lu TetR, 1205LuTetR siGRM1-1, or 1205 Lu TetR siGRM1-9 treated with either plain media or 10 ng/mL doxycycline for 7 days and assayed for GRM1, phosphorylated and total ERK1/2, phosphorylated AKT (S473), phosphorylated AKT2, and α-tubulin expression. The blots were scanned and the intensity of the protein bands analyzed with Optiquant Software (PerkinElmer Life Sciences).

MTT assays were conducted as described (6) with 1 × 10³ cells per well of C8161 pVgRXR siGRM1 clones A10, and C8161 pVgRXR siGRM1 A13, or C8161 pVgRXR. The conditions were no treatment (NT), vehicle (DMSO) or 1, 5, and 10 μmol/L of PonA for 7 days. Absorbance was determined on days 0, 3, and 7 using a Tecan Plate reader (Infinite 200 Tecan USA).

C8161 pVgRXR siGRM1 A13 cells and vector control C8161 pVgRXR cells were injected at 10⁶ cells per site in the dorsal flanks of 5- to 6-week-old athymic nude mice. When the tumor volumes reached approximately 10 to 20 mm³ as measured with a vernier caliper, mice were randomly divided into 2 groups with similar tumor volumes with 6 mice per group. One group was treated with vehicle (olive oil; Veh) and the other one with PonA (10 mg/kg). Mice were treated twice a week with vehicle or PonA administered via intraperitoneal injection as described (16) and measured once a week with a vernier caliper. Tumor volumes were calculated as described (17). All tumor-bearing mice were euthanized after 37 to 42 days due to tumor burden in the control groups.

1205 Lu TetR siGRM1-9, 1205 Lu TetR siGRM1-1, or UACC903 TetR siGRM1-8 and UACC903 TetR siGRM1-11 cells were injected as 10⁶ cells per site in the dorsal flanks of 5- to 6-week-old athymic nude mice. When the tumor volumes reached approximately 10 to 20 mm³ as measured with a vernier caliper, mice were divided into no treatment (NT) or doxycycline (Dox) groups with similar tumor volumes in each group with 6 mice per group. A 0.1% doxycycline and 1% w/v sucrose solution was provided to the animals and replaced twice weekly in the Dox treatment groups as previously described (7). The tumor volume was measured once a week, and all tumor-bearing mice euthanized when the tumor burden in the control groups approached maximum permitted levels.

Immunohistochemical staining on excised tumor xenografts from C8161 pVgRXR and C8161 pVgRXR siGRM1 A13 PonA or vehicle-treated controls, 1205 Lu TetR and 1205 Lu TetR siGRM1-9 doxycycline-treated and not-treated controls to detect changes in the number of apoptotic and proliferating cells (cleaved caspase-3 and Ki-67, respectively) was conducted by Tissue Analytical Services at the Cancer Institute of New Jersey (New Brunswick, NJ). The number of stained cells was quantified with a digital Aperio ScanScopeGL system and ImageScope software (v 10.1.3.2028; Aperio Technologies Inc.) according to the manufacturer's protocol with modifications as described (18).

Statistics analysis was calculated using Microsoft Excel with 2-sample t tests with statistical significance set at P < 0.05.

GRM1-positive C8161 human melanoma cells were transfected with a plasmid construct encoding a heterodimeric receptor consisting of the ecdysone receptor (EcR) and the mammalian homologue of the ultraspiracle protein and the retinoid X receptor (RXR; pVgRXR). Cells expressing the receptor were then transfected with a plasmid encoding GRM1 siRNA (siGRM1). This construct also contains 5 copies of the ecdysone response element (EcRE) which are upstream of a minimal promoter and which can drive expression of siGRM1 in the presence of the inducer, an analogue of ecdysone, PonA (14, 15). We showed that in the presence of the inducer, 10 μmol/L PonA, GRM1 expression was diminished in several clones with examples of 2 such clones shown (Fig. 1A), whereas cells containing the RXR receptor but lacking the siGRM1-RNA, C8161 pVgRXR did not show any decrease in levels of GRM1 expression (Fig. 1A). Quantitation of the intensity of the Western blots indicate an approximately 40% decrease in C8161 pVgRXR siGRM1 A13 and an approximately 50% decrease in C8161 pVgRXR siGRM1 A10 (Fig. 1B).

For the tetracycline-inducible system, GRM1-positive UACC903 and 1205 human melanoma cells were infected with a lentiviral construct to ensure high expression levels of the TetR. The TetR-positive cells were then transfected with si-GRM1-RNA and treated with 10 ng/mL doxycycline to induce expression of siGRM1. Several independent clones were isolated and examples of some of the clones are shown. 1205 Lu TetR siGRM1 clones 1 and 9 and UACC903 TetR clones 8 and 11 exhibited decreased GRM1 expression compared with 1205 Lu TetR and UACC903 TetR cells that did not display any decrease in GRM1 expression after treatment with doxycycline (Fig. 1C). Quantitation of the intensity of the no treatment and doxycycline-treated samples showed that in the 1205 Lu TetR siGRM1 cells, clones 1 and 9 exhibited an approximately 90% decrease in GRM1 expression, whereas (Fig. 1D, left) UACC903 TetR siGRM1 clones 8 and 11 exhibited an approximately 90% and 80% decrease in GRM1 expression, respectively (Fig. 1D, right).

We have shown previously that when the GRM1 receptor is rendered nonfunctional by treatment with a competitive or noncompetitive GRM1 antagonist, it results in suppression of the proliferation in vitro, in several GRM1-expressing human melanoma cell lines (6, 19). We conducted MTT cell viability/proliferation assays on clones from the ecdysone-regulated siRNA expression system; we showed that treatment with the inducer, PonA, resulted in a 15% to 30% decrease in C8161 pVgRXR siGRM1 cells, whereas control C8161 pVgRXR cells did not show any modulation in cell growth (Fig. 2A). Furthermore, we also showed that a reduction in viable cell number, at least in part, could be attributed to an increase in the apoptotic cell population as assessed by a rise in the levels of an apoptotic marker, the cleaved form of PARP (Fig. 2B).

Possible alterations in MAPK signaling cascade by suppression of GRM1 in human melanoma cells were assessed. Treatment of C8161 pVgRXR siGRM1 A13 with the inducer, PonA, resulted in a decrease in the levels of activated/phosphorylated ERK1/2 in comparison with vector control C8161 pVgRXR (Fig. 3A). Quantification of the intensities of phosphorylated form of ERK1/2 and normalized to the no treatment or vehicle controls showed a 40% decrease in levels of the activated ERK1/2 in C8161 pVgRXR siGRM1 A13 lysates (Fig. 3B). Using the doxycycline-inducible system, we also observed a reduction in ERK1/2 phosphorylation in UACC903 TetR siGRM1 clones induced with 10 ng/mL doxycycline, which was not observed in UACC903 TetR cells under similar conditions (Fig. 3C). Quantitation of the Western blots comparing phosphorylated ERK1/2 with total ERK1/2 showed an approximately 50% and 75% reduction in the intensity in UACC903 TetR siGRM1 clones 8 and 11, respectively (Fig. 3D). In 1205 Lu TetR siGRM1 clones, we also observed a reduction in phosphorylated ERK1/2 with clone 9 showing greater suppression than clone 1(Fig. 3E). Quantitation of the Western blots comparing phosphorylated ERK1/2 with total ERK1/2 showed an approximately 27% and 64% reduction in intensity in the 1205 Lu TetR siGRM1 clones 1 and 9, respectively (Fig. 3F).

We have previously shown that the PI3K/AKT pathway functions downstream of Grm1 and also identified the Akt2 isoform to be the mediator of Akt phosphorylation in our system (20). Here, we investigated whether there are changes in AKT phosphorylation with siRNA-mediated suppression of GRM1 expression. Our results indicate that suppression of GRM1 expression by siRNA can inhibit the phosphorylation and activation of AKT in UACC903 human melanoma cells (Fig. 4A). Similar results were observed in siGRM1-1205 Lu human melanoma cell lines (Fig. 4C). Furthermore, we showed that AKT phosphorylation is mediated by the AKT2 isoform (Fig. 4A and C). Quantitation of the Western blots comparing phosphorylated AKT with total AKT showed an approximately 40% and 50% reduction in the intensity in UACC903 TetR siGRM1 clones 8 and 11, respectively (Fig. 4B, left), whereas 1205 Lu TetR siGRM1 clones 1 and 9 showed an approximately 30% and 40% decrease in clones 1 and 9, respectively (Fig. 4D, left). Comparison of AKT2 phosphorylation normalized to total AKT showed an approximately 50% and 15% reduction in the UACC903 TetR siGRM1 clones 8 and 11, respectively (Fig. 4B, right), whereas 1205 Lu TetR siGRM1 clones 1 and 9 had an approximately 50% and 40% decrease, respectively (Fig. 4D, right).

Next, we conducted in vivo xenograft experiments with vector control C8161 pVgRXR and C8161 pVgRXR siGRM1 A13 cells (Fig. 5A and B). The cells were inoculated into the dorsal flanks of nude mice and when the tumor volumes reached approximately 10 to 20 mm³, the mice were randomly divided into 2 groups, either treated with vehicle (olive oil) or 10 mg/kg of PonA in olive oil by intraperitoneal injections twice weekly as described (16, 21). We showed that in the C8161 pVgRXR siGRM1 A13 xenografts treated with PonA, the tumors appeared to progress at a slower rate compared with those treated with the vehicle and resulted in an approximately 45% (P < 0.005) decrease in tumor volume. In contrast, in the control C8161 pVgRXR, there was no suppression of tumor growth when treated with PonA; in fact, we observed slightly faster growth in comparison with vehicle-treated ones (Figs. 5A and B). We also conducted xenograft studies with cells harboring tetracycline-inducible siRNA. 1205 Lu TetR siGRM1 clones 1 and 9 or UACC903 TetR clones 8 and 11. Mice were randomly divided into no treatment and treatment groups when the tumor volumes reached approximately 10 to 20 mm³. A 0.1% doxycycline and 1% sucrose w/v solution was administered in the drinking water in the Dox treatment groups. The data indicate that suppression of GRM1 in 1205 Lu cells and siGRM1 cells treated with doxycycline resulted in approximately 50% (P < 0.005) and 60% (P < 0.005) reduction in tumor volumes in clones 1 and 9, respectively, compared with those in the no treatment control groups (Fig. 5C and D). Similarly, in UACC903 TetR siGRM1 cells, inhibition of GRM1 expression resulted in approximately 56% (P < 0.05) and 47% (P < 0.05) reduction in tumor growth in vivo in clones 8 and 11, respectively (Fig. 5E and F).

Tumors excised from C8161 pVgRXR, C8161 pVgRXR siGRM1 A13 PonA, or vehicle-treated samples and 1205 Lu TetR and 1205 Lu TetR siGRM1-9 doxycycline-treated or no treatment controls were analyzed by immunohistochemistry for the expression of the apoptosis marker, cleaved caspase-3, and the proliferation marker, Ki-67. C8161 pVgRXR–derived tumors did not show any significant increase in cleaved caspase-3 or a decrease in Ki-67 (Fig. 6A). C8161 pVgRXR siGRM1 A13–derived tumors treated with PonA exhibited a statistically significant increase in cells positive for cleaved caspase-3 over vehicle-treated samples (P < 0.05; Fig. 6B, left). Analysis of the same samples showed a statistically significant decrease in Ki-67 expression in the PonA-treated samples over vehicle-treated samples, (P < 0.001; Fig. 6B, right). Using the doxycycline-regulated siRNA system, no differences were observed in cleaved caspase-3 and Ki-67 in the 1205 Lu TetR vector controls after doxycycline treatment (Fig. 6C). In the 1205 Lu TetR siGRM1-9–derived tumors, treatment with doxycycline led to a statistically significant increase in cells positive for cleaved caspase-3 over the not-treated controls with P < 0.05 (Fig. 6D, left). Meanwhile, a statistically significant decrease in Ki-67 cells was detected in the doxycycline-treated 1205 Lu TetR siGRM1-9 tumors than in the no treatment controls with P < 0.001 (Fig. 6D, right). These results showed that in vivo suppression of GRM1 expression in these human melanoma cells has both antiproliferative and proapoptotic effects.

We have previously reported that targeted Grm1 expression to melanocytes was sufficient to induce spontaneous melanoma development in vivo; subsequently, we also detected GRM1 expression in a subset of human melanoma cell lines and biopsies suggesting that GRM1 may be involved in melanomagenesis (2, 3, 6). In addition, 2 other metabotropic glutamate receptors have recently been documented to be involved in melanoma pathogenesis. Overexpression of Grm5 (12) and mutations in GRM3 (13) make this particular group of receptors and glutamate signaling attractive candidates for understanding the onset and progression of this deadly disease.

Gene silencing by siRNAs (22) has been used to silence the expression of specific genes to study their roles in different cell types and in various organisms. Inducible gene expression systems have also been developed to regulate expression in a temporal and quantitative manner to aid in the study of gene function (21). These regulatable expression systems rely on small molecules that serve as inducers to modify synthetic transcription factors, which regulate the expression of a target gene (21). The tetracycline operon–based, tetracycline-inducible system (7, 23, 24) and the nonmammalian steroid–based, ecdysone-inducible system (15, 16, 25) have been shown to be both reversible and efficient in regulating the expression of various mammalian genes. These inducible systems are especially critically important when siRNAs are used in inhibiting expression of genes crucial for cell or organism survival, as silencing occurs only in the presence of the inducer (14, 26). We and others have shown that the sustained expression of Grm1 in mouse melanocytes is required for the maintenance of transformed phenotypes in vitro and tumor progression in vivo (7, 8). In our attempts to inhibit GRM1 expression in human melanoma cells, we have previously used constitutively expressed GRM1 specific siRNAs with unfavorable consequence as the GRM1 knockout cells exhibited a dormancy-like state before dying (unpublished observations). We selected the ecdysone/Pon A and the tetracycline-inducible gene expression systems to induce siRNA expression targeted to GRM1 in human melanoma cell lines.

It has been shown that GRM1 activation is coupled to the extracellular signal-regulated kinase (ERK1/2), a component of the classical MAPK pathway through G-protein and kinase-dependent mechanisms in the neuronal cells (27). Activation of ERK1/2 by phosphorylation on tyrosine and threonine residues has a pivotal role in intracellular signaling and can mediate cellular processes such as cell proliferation, invasion, metastasis, survival angiogenesis, and apoptosis (28, 29). Our group has shown that similar to the neuronal system, stimulation of GRM1 by its agonist, l-quisqualate, leads to the activation of ERK1/2 in murine or human melanoma cells (6, 7, 30). The specificity of GRM1-mediated activation of ERK1/2 was shown by preincubation of the cells with GRM1-antagonists, Bay 36-7620 or LY-367385, which abolished ERK1/2 phosphorylation (6, 7, 30). Activated MAPK pathway has also been detected with other metabotropic glutamate receptors in melanoma. The overexpression of Grm5 in mouse melanocytes was found to result in ERK1/2 activation (12), whereas mutations in GRM3 were found to activate MEK1/2 (13) indicating that the MAPK pathway has a fundamental role in melanomas linked to glutamate receptors. Stimulation of the MAPK pathway is indeed a classical event in melanoma due to mutations in BRAF and RAS (31, 32). In this report, our results indicate that suppression of GRM1 expression in human melanoma cells results in reduced activation of the MAPK pathway. These results suggest that in melanoma cells, MAPK activation can also result from upstream receptor-mediated activation events such as the activation of GPCRs including GRM1.

In addition to the suppressed MAPK pathway stimulation observed, we showed that suppressed GRM1 expression by siRNA leads to inhibition of another major signaling cascade, PI3/AKT. The PI3K/AKT pathway plays critical roles in cell survival, proliferation, and apoptosis through PTEN loss or aberrant AKT activation (20, 33, 34). Our earlier work in mouse melanocytes showed that Grm1 activation by its agonist l-Quisqualate could result in AKT activation and phosphorylation. The specificity of this activation was confirmed with the Grm1-specific antagonist Bay-36-7620, which blocked the activation and phosphorylation of AKT (20). Moreover, we showed that the AKT2 isoform mediated this activation (20). Here, we showed that a decrease in GRM1 expression by siRNA resulted in the suppression of AKT phosphorylation and distinctively, the phosphorylation of AKT2. This suppression of AKT2 phosphorylation has significant implications, as it has been shown to have proinvasive properties in melanoma cells (35).

Despite the lack of specific GRM1 antagonists with clinical applications, our group has used Rilutek (riluzole), an U.S. Food and Drug Administration (FDA)-approved drug for the treatment of amyotropic lateral sclerosis (36, 37) in clinical trials for patients with melanoma (38, 39). Riluzole inhibits the release of glutamate from intracellular to extracellular environments (40, 41). GPCRs with oncogenic activity are known to create autocrine/paracrine loops to maintain receptor activation (42–44). Inhibition of glutamate release by riluzole in GRM1-expressing cells disrupts glutamate receptor signaling and thus functions as an antagonist to the receptor (6). Results from our phase 0 and II trials strongly suggest that riluzole monotherapy has modest antitumor activity (38, 39); however, combining it with other anticancer agents may lead to additive or synergistic responses as shown in our preclinical studies (45). On the basis of these results, perhaps, it is not surprising that modulation of GRM1 expression results in concurrent suppression of activities of 2 major signaling pathways known to be critical in melanomagenesis.

No potential conflicts of interest were disclosed.

Conception and design: J. Wangari-Talbot, J.S. Goydos, S. Chen

Development of methodology: J. Wangari-Talbot, S. Chen

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Wangari-Talbot, B.A. Wall, J.S. Goydos, S. Chen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Wangari-Talbot, B.A. Wall, J.S. Goydos, S. Chen

Writing, review, and/or revision of the manuscript: J. Wangari-Talbot, B.A. Wall, J.S. Goydos, S. Chen

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Chen

Study supervision: J.S. Goydos, S. Chen

This work was supported by the grants from NIH GM66338 and R01CA124975-02S1 (to J. Wangari-Talbot), New Jersey Commission for Cancer Research 09-1143-CCR-E0 and NIH R01CA74077 (to S. Chen), and NIH R01CA124975 (to J.S. Goydos).

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.