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. 2012 May 1;128(2):517–523. doi: 10.1093/toxsci/kfs165

The Insecticide Synergist Piperonyl Butoxide Inhibits Hedgehog Signaling: Assessing Chemical Risks

Jiangbo Wang *, Jiuyi Lu *, Robert A Mook Jr *, Min Zhang *, Shengli Zhao *, Larry S Barak †, Jonathan H Freedman ‡, H Kim Lyerly §, Wei Chen *,1
PMCID: PMC3493191  PMID: 22552772

Abstract

The spread of chemicals, including insecticides, into the environment often raises public health concerns, as exemplified by a recent epidemiologic study associating in utero piperonyl butoxide (PBO) exposure with delayed mental development. The insecticide synergist PBO is listed among the top 10 chemicals detected in indoor dust; a systematic assessment of risks from PBO exposure, as for many toxicants unfortunately, may be underdeveloped when important biological targets that can cause toxicity are unknown. Hedgehog/Smoothened signaling is critical in neurological development. This study was designed to use novel high-throughput in vitro drug screening technology to identify modulators of Hedgehog signaling in environmental chemicals to assist the assessment of their potential risks. A directed library of 1408 environmental toxicants was screened for Hedgehog/Smoothened antagonist activity using a high-content assay that evaluated the interaction between Smoothened and βarrestin2 green fluorescent protein. PBO was identified as a Hedgehog/Smoothened antagonist capable of inhibiting Hedgehog signaling. We found that PBO bound Smoothened and blocked Smoothened overexpression–induced Gli-luciferase reporter activity but had no effect on Gli-1 downstream transcriptional factor–induced Gli activity. PBO inhibited Sonic Hedgehog ligand–induced Gli signaling and mouse cerebellar granular precursor cell proliferation. Moreover, PBO disrupted zebrafish development. Our findings demonstrate the value of high-throughput target-based screening strategies that can successfully evaluate large numbers of environmental toxicants and identify key targets and unknown biological activity that is helpful in properly assessing potential risks.

Key Words: piperonyl butoxide, Smoothened antagonist, Hedgehog signal transduction pathway, high throughput screening, chemical risk assessment.

Piperonyl butoxide (PBO) was developed prior to World War II during America’s attempt to maintain its strategic reserves of 
pyrethrum insecticides and develop human-safe alternatives. There are over 1500 products containing PBO (Daiss, 2010), including products directly applied on human skin with PBO concentration up to 4% (Meinking et al., 2002), which is about 118µM. Widely used as a pesticide synergist, PBO (Fig. 1A) enhances pyrethrum efficacy by inhibiting insect cytochrome P450– mediated detoxification (Hodgson and Levi, 1998). PBO/pyrethrum combinations are widely applied both in agriculture and in homes as aerosols and powders, placing PBO among the top 10 chemi cals detected in indoor dust (Hodgson and Levi, 1998; Rudel
et al., 2003).

FIG. 1.

FIG. 1.

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PBO regulates the Smo-mediated intracellular distribution of βarr2-GFP. (A) PBO structure. Confocal images of U2OS cells stably expressing 
(B) βarr2-GFP alone or (C–E) βarr2-GFP with Smo-633. Cells were treated for 6 h with dimethyl sulfoxide (DMSO) (C), 5µM cyclopamine (Cyc) (D), or 12.5µM PBO (E), n = 3. Scale bar: 10 µm.

The spread of chemicals, including insecticides, into the environment often raises public health concerns. A recent human epidemiologic study involving a population of pregnant women in the United States found a significant association between PBO collected in their personal air during pregnancy and delayed mental development in the children (Horton et al., 2011). The children born to mothers who were more highly exposed to PBO during their pregnancy scored 3.9 points lower on the Mental Developmental Index compared with those with lower exposures, a degree of impairment often seen with chronic lead exposure, a recognized environmental hazard (Lanphear et al., 2005).

The systematic assessment of risks from chronic toxicant exposure can be underdeveloped when the biological targets capable of causing toxicity are unknown and often rely on detecting phenotypic or behavioral changes in small animal models. High-throughput in vitro technologies, often used in drug discovery, provide potential solutions for identifying elusive toxicity-associated biological targets and, moreover, have the potential to supplant animal-based toxicology screens. We tested this idea by screening a toxicant library of 1408 chemicals against a highly relevant biological target based on the epidemiologic observations. We discovered that PBO binds the seven-transmembrane receptor Smoothened (Smo) and inhibits Hedgehog signaling, a critical regulator of stem cell proliferation, cancer, and central nervous system development (Ingham and McMahon, 2001; Marti and Bovolenta, 2002; Ruiz i Altaba et al., 2002; Stecca and Ruiz i Altaba, 2005).

MATERIALS AND METHODS

Reagents. The Biomolecular Screening Branch of the National 
Toxicology Program provided the Chemical Toxicant Library of 1408 compounds that included PBO (Xia et al., 2008). PBO was purchased from 
AccuStandard (> 97% purity by gas chromatography). PBO purchased from Sigma-Aldrich (technical grade, 90% purity) gave similar results in the primary assay. The data reported within were generated with PBO from AccuStandard, the material of higher purity. Permethrin (PEM) and cyclopamine were purchased from Sigma and Toronto Research Chemicals, respectively. βarrestin2 green fluorescent protein (βarr2-GFP), wild-type Smo, Smo-663 mutant, and Gli-luciferase reporter have been previously described (Chen et al., 2004; Wang et al., 2010). Gli-1 construct was provided by Dr Rune Toftgard of Karolinska Institute, Sweden. F-59 and 4D9 antibodies were obtained from the Developmental Studies Hybridoma Bank of the University of Iowa.

Animals and whole-mount immunostaining. Wild-type zebrafish were raised and bred in a water recirculation system at 28.5°C on a 14-h light/10-h dark cycle. Whole-mount immunostaining was performed as previously described (Bader et al., 1982).

Primary assay–automated high-throughput screening protocol. The screening protocol to identify antagonists of Smo used screening methodology previously reported to identify Smo agonist (Wang et al., 2010), with the exception that no cyclopamine pretreatment occurred prior to addition of the test compounds.

Binding of overexpressed Smo in U2OS cells. Cells in 24-well plates were fixed with 4% (v/v) formaldehyde/PBS for 20 min at room temperature (RT) followed by incubation for 2 h at RT in binding buffer (Hank's Balanced Salt Solution [HBSS] salt without Ca2+ and Mg2+) containing a range of concentrations from 0 to 50nM [3H]cyclopamine for saturation binding; or 25nM [3H]cyclopamine plus cyclopamine (range from 0 to 10µM), PBO (range from 0 to 31.6µM), or PEM (range from 0 to 31.6µM) for competitive binding. Cells were then washed with HBSS (without Ca2+ and Mg2+), and the bound [3H]cyclopamine was extracted with 200µl of 0.1 N NaOH. The extracts were neutralized with 200µl of 0.1 N HCl and then added to scintillant for counting. Specific binding over baseline was normalized to the maximal binding [3H]cyclopamine. Data were analyzed with GraphPad Prism by nonlinear regression.

Gli-luciferase reporter assay and primary neuronal granular cell precursors [ 3 H]thymidine proliferation assay. The Gli-luciferase assay was conducted in NIH3T3 cells and Shhlight-2 cells. Granular cell precursors (GCPs) obtained from 7-day-old mouse cerebellum were used in the [3H]thymidine proliferation assay. Both assays were performed as previously described (Wang et al., 2010).

RESULTS

PBO Inhibits Intracellular Aggregation of Smo With βarr2-GFP

The high-throughput assay exploited our discovery that activated wild-type Smo or Smo-633 binds βarr2-GFP and changes its cellular distribution (Chen et al., 2004; Wang et al., 2010). βarr2-GFP distributes homogenously throughout the cytoplasm when expressed in cells without activated Smo or Smo-633 (Fig. 1B). Overexpression of Smo-633 causes βarr2-GFP to change from a homogeneous distribution to intracellular vesicles/aggregates (Fig. 1C). Addition of a Smo antagonist, such as cyclopamine, inhibits the aggregation of Smo-633 with βarr2-GFP as demonstrated by the disappearance of intravesicular aggregates (Fig. 1D). Upon screening 1408 toxicants at a single concentration of 12.5µM, only PBO inhibited βarr2-GFP intravesicular aggregates in cells (Fig. 1E) similar to that observed with cyclopamine. A similar response was obtained upon PBO exposure to U2OS cells expressing βarr2-GFP and wild-type Smo (Supplementary fig. 1). Although the insecticidal toxicity of PEM is enhanced by PBO, PEM had no effect on the distribution of βarr2-GFP with either Smo-633 or wild-type Smo in cells stably overexpressing βarr2-GFP (Supplementary fig. 2). To control for receptor specificity and to rule out nonspecific mechanisms, we examined whether PBO could act as an antagonist at a different seven-transmembrane receptor in the cells. Neither cyclopamine nor PBO prevented βarr2-GFP aggregation in response to vasopressin2 receptor (V2R) activation by the agonist arginine vasopressin in cells transfected with V2R and βarr2-GFP (Supplementary fig. 3). These data suggest that the mechanism of inhibition is Smo receptor specific and rule out mechanisms of inhibition that are nonspecific.

PBO Displaces [3H]cyclopamine in Cells Overexpressing Smo

To investigate the binding of PBO to Smo, we established a [3H]cyclopamine binding assay in U2OS cells overexpressing wild-type Smo. The affinity (Kd) of [3H]cyclopamine for Smo is 12.4 ± 4.2nM (Supplementary fig. 4). Next, we tested the ability of PBO to competitively displace [3H]cyclopamine from Smo. Cyclopamine completely displaced 25nM [3H]cyclopamine from Smo (defined as an efficacy of 1.00) with an affinity (Ki) of 14.3 ± 0.5nM (Fig. 2). The Ki and efficacy for displacement for PBO were 855 ± 12 nM and 0.53 ± 0.03, respectively. As expected, PEM did not compete [3H]cyclopamine from Smo (Fig. 2).

FIG. 2.

FIG. 2.

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PBO competitively displaces [3H]cyclopamine binding to Smo. Competitive binding of [3H]cyclopamine with Smo antagonists was performed in fixed U2OS cells, as described in Materials and Methods section. Data were normalized to the maximal binding of [3H]cyclopamine over baseline. The displacement data were analyzed by fitting to a one-site competition curve using GraphPad Prism. Data were acquired in triplicate from three independent experiments and are presented as the mean ± SEM.

PBO Inhibits Smo-Induced Gli-luciferase Reporter Activity but Has No Effect on Downstream Gli-1 Induced Gli Activity

It has been reported that a Smo antagonist can block Hedgehog pathway activity produced by Smo overexpression (Kim et al., 2010). As expected, 10µM cyclopamine or 10µM PBO significantly inhibited the Smo-induced Gli-luciferase reporter activity when Smo was overexpressed in NIH3T3 cells (Fig. 3A). To further confirm PBO specificity to Smo, we overexpressed Gli-1, a Smo downstream transcriptional factor, in NIH3T3 cells and found that neither cyclopamine nor PBO had any significant effect on Gli-1–induced Gli-luciferase reporter activity (Fig. 3B). These results indicate that PBO inhibits the Hedgehog signaling pathway by directly inhibiting Smo, rather than inhibiting the downstream transcriptional factor Gli-1.

FIG. 3.

FIG. 3.

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PBO inhibits Smo-induced Gli-reporter activity but has no effect on Gli-1–induced Gli activity. NIH3T3 cells were transfected with Gli-luciferase reporter and control renilla pRL-TK (Promega) plasmids, together with empty vector, Smo (A) or Gli-1 (B). The transfected cells were incubated for 48 h before treatment with DMSO, 10µM cyclopamine (Cyc), or 10µM PBO for 20–22 h. Data are presented as mean ± SEM with n = 3. The statistical significance was analyzed by two-tailed Student’s t-test with p < 0.05 (alpha = 0.05) defined as significant.

PBO Inhibits Gli-luciferase Reporter Activity and Proliferation of Mouse Cerebellar GCPs

Sonic Hedgehog (Shh) binding to Patched relieves Patched inhibition of Smo and results in Gli activation (Taipale et al., 2000). A Gli-luciferase reporter assay using Shhlight-2 cells is commonly used to measure Smo activation of Gli (Chen et al., 2002). Utilizing this assay, both cyclopamine and PBO inhibited Shh-induced Gli activation with IC50 values of 0.39 ± 0.07µM and 1.62 ± 0.7µM, respectively (Fig. 4A).

FIG. 4.

FIG. 4.

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PBO inhibits Gli-reporter activity and GCP proliferation. (A) Gli-luciferase response in Shhlight-2 cells treated for 30 h with 20% Sonic Hedgehog conditioned media (Shh) in the presence of cyclopamine (Cyc), PBO, or PEM. (B) GCP cells were treated for 48 h with Shh and Cyc, PBO, or PEM. Cells exposed to [3H]thymidine for 16 h were measured for [3H]thymidine incorporation. Data were fit using GraphPad Prism (mean ± SEM, n = 3).

Cerebellar GCPs are abundant in the brain. The proliferation of these neuron precursors in vivo requires Hedgehog/Smo pathway signaling (Wechsler-Reya and Scott, 1999). We used a mouse GCP proliferation assay (Wang et al., 2010) to assess the growth-inhibiting effects of cyclopamine and PBO. Relative to Shh stimulation alone, treatment with PBO resulted in a dramatic decrease in GCP proliferation (IC50 = 3.35 ± 0.8µM). The response was similar to the maximal response following cyclopamine exposure (IC50 = 0.49 ± 0.16µM) (Fig. 4B). As expected, PEM did not inhibit Shh-induced Gli-activation (Fig. 4A) or GCP proliferation (Fig. 4B).

To further examine PBO at a functional level, we investigated the ability of the Smo agonist SAG to rescue PBO block of Hedgehog signaling in the presence of Shh ligand in both Shhlight-2 cells and GCP cells. In this experiment, we expect that SAG would rescue Shh-activated Smo signaling if signaling was blocked by a true Smo antagonist. Indeed, while either 10µM cyclopamine or 10µM PBO can inhibit Shh-activated Gli activity and GCP proliferation, such inhibition can be overcome by 0.3µM SAG (Supplementary fig. 5).

PBO Disrupts Zebrafish Embryo Development

To assess the inhibitory effect of PBO on Hedgehog/Smo signaling in vivo, we utilized an established zebrafish embryo model. In this model, disruption of Hedgehog-controlled development of slow muscle fibers and its origin muscle pioneer cells lead to a characteristic abnormal somite phenotype (Wilbanks et al., 2004). Treatment of zebrafish embryos with PBO or cyclopamine disrupted the normal V-shaped somite phenotype (Fig. 5A–C) and the arrangement of slow muscle fibers (Fig. 5D–F). Muscle pioneer cells express Engrailed protein (Hatta et al., 1991). Staining of Engrailed was markedly decreased in both cyclopamine- and PBO-treated embryos (Fig. 5G–I). The abnormal phenotype caused by PBO and cyclopamine supports the finding that PBO disrupts Hedgehog signaling in vivo.

Fig. 5.

Fig. 5.

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Effects of PBO on developing zebrafish embryos. Embryos treated for 20 h in water containing 0.1% DMSO (A, D, and G), 5µM cyclopamine (Cyc) (B, E, and H), or 25µM PBO (C, F, and I) 4 h after fertilization. Images A, B, and C are lateral views of whole zebrafish; D, E, and F are F59 anti-myosin antibody staining of slow muscle fibers; and G, H, and I are 4D9 antibody staining of Engrailed (n = 3).

DISCUSSION

The use of pesticides with PBO as a synergist has resulted in widespread presence of PBO in surface water (Woudneh and Oros, 2006) and indoor dust (Rudel et al., 2003). Upon exposure, PBO is well absorbed from oral, inhalation, and topical routes of exposure. In rats, maximal plasma concentrations of PBO occurred 5 h after oral administration at a dose of 250 mg/kg, with higher concentrations in brain (23 µg/g), testis (19 µg/g), liver (33 µg/g), and adipose tissue (201 µg/g) than plasma (7 µg/g). After 24 h, PBO was 
detected in the plasma, but none remained in the brain, liver, and kidney. Accumulation of PBO in adipose tissue was remarkable, with PBO reaching a peak concentration (800 µg/g) after 12 h and still observed after 96 h (Kimura et al., 1985). Consistent with these distribution studies, PBO has a high logP (4.95) (Daiss, 2010), thus supporting its propensity to distribute into lipid and tissues from plasma. A study using intratracheal administration of PBO to rats found initially high levels of biliary excretion of PBO followed by a prolonged period of hepatic elimination, suggesting that PBO can be absorbed from lung and excreted by liver into bile (Fishbein et al., 1972). A recent study in humans tested 5-day dermal exposure to PBO (dosed at 100 µg/cm2) and determined that 8.9% of the compound was absorbed through skin (Ross et al., 2011). PBO was also detected in 75.2% of the personal air samples of 725 pregnant women (Horton et al., 2011). In a separate study of 297 newborns, PBO was detected at concentrations at or above 70 pg/ml in 36% of the cord serum samples (Neta et al., 2010). These studies suggest that environmental exposure of PBO to humans and animals results in acute and chronic exposure to the compound.

The epidemiologic association of maternal PBO exposure during gestation and later defects of mental function in their children suggests a potential neurotoxic effect of PBO in humans (Horton et al., 2011). However, the molecular mechanism responsible for this finding is unknown. Our in vitro and in vivo studies indicate that concentrations of PBO that produce sufficient Smo antagonism can negatively impact biological processes that require Hedgehog signaling. The duration and level of PBO exposure during key developmental events in utero may offer a potential mechanism of the defect found in children. Further studies are required to test the mechanism.

Although we cannot conclude from our studies that the levels of PBO that are typically found in the environment will have deleterious consequences to public health, it is clear that the concentration of PBO found in many of the over 1500 products far exceed the concentration required to inhibit Hedgehog signaling in our assays. For example, products directly applied on human skin contain up to 4% PBO (Meinking et al., 2002). Additional studies are needed to adequately compare the levels of exposure of internal tissues to PBO, to the concentration of PBO that inhibits Hedgehog signaling in our assays. Given the propensity of PBO to distribute into tissues, it is difficult to correlate the plasma concentrations observed in limited studies in humans with inhibition data in our study. For example, Neta et al. (2010) reported levels equal or greater than 70 pg/ml PBO in cord blood (corresponding to 0.21nM), a concentration lower than the concentrations of PBO used in our studies. Tissue concentrations of PBO in the newborn were not reported in the epidemiologic study of Horton et al. (2011). In animal studies, the concentrations of PBO in both plasma and tissues vary depending on the dose and route of administration. In the study of Kimura et al. (1985), the concentrations of PBO observed in tissues are comparable or higher than the IC50 values observed in our assays and would be anticipated to be high enough to negatively impact Hedgehog signaling.

A variety of studies, including genetic knockout studies and studies of Hedgehog signaling inhibitors, provide insights into in vivo effects anticipated by inhibition of Hedgehog signaling. Defects in brain and spinal cord, axial skeleton, and limb structures are observed in Shh knockout mice (Chiang et al., 1996), and facial and limb defects are often observed in developing animals when administered Hedgehog signaling inhibitors (Lipinski and Bushman, 2010). In neurodevelopment, Hedgehog signaling is crucial in regulating many processes in the development of the nervous system, including differentiation of the midbrain and ventral forebrain, and proliferation of hippocampal neuronal precursor cells (Lai et al., 2003; Ruiz i Altaba et al., 2002; Stecca and Ruiz i Altaba, 2005). Shh is required for cell proliferation in the mouse forebrain subventricular zone stem cell niche and for the production of the new olfactory interneurons, inhibition of Shh signaling with cyclopamine decreases cell proliferation in subventricular zone (Palma et al., 2005). In addition to its role in neurodevelopment, Hedgehog plays a critical role in patterning of the face (Chiang et al., 1996; Lipinski and Bushman, 2010). In bone development, oral dosing of 10-day-old mice with a small-molecule Smo inhibitor for as little as 2–4 days caused dramatic and permanent defects in bone structure, including malformation of the epiphysis and growth plate (Kimura et al., 2008).

PBO has been assigned toxicity Category IV by all routes of exposure by the U.S. Environmental Protection Agency (EPA) (Daiss, 2010). Category IV is generally considered practically nontoxic, not an irritant, although new data requirements under 40 CFR part 158 require additional neurotoxicity studies. According to EPA documents, the U.S. EPA believes the guideline studies are inadequate in their assessment of behavior effects and do not use optimal methods to evaluate the potential toxicity to the nervous tissue structure and function. The objective of additional neurotoxicity battery testing is to evaluate the incidence and severity of functional and/or behavior effects, the level of motor activity, and the histopathology of the nervous system. Acute and subchronic neurotoxicity studies are required. Subchronic study is intended to detect possible effects resulting from repeated or long-term exposure. Although not previously recognized as a Hedgehog pathway inhibitor, some of the toxicity reported appear consistent with effects on the Hedgehog pathway (Kimura et al., 2008; Tanaka et al., 1994, 2009). For example, PBO has been reported to have adverse effects on neurobehavioral parameters in mice fed a PBO containing diet in a two-generation toxicity study (Tanaka et al., 2009). Specifically, cliff avoidance (indicative of development of consciousness), surface righting (indicative of development of coordinated movements), and time to olfactory orientation (indicative of development of the sense of smell) were all delayed in the F1-generation offspring during the early lactation period in a sex-related manner. In developmental toxicity studies, PBO exposure was associated with oligodactyly, malformation of forelimbs, indicating teratogenic effects of PBO in mice (Tanaka et al., 1994). Defects in limb are observed in Hedgehog knockout mice (Chiang et al., 1996). Bone defects are observed in juvenile mice treated with a Smo antagonist (Kimura et al., 2008). Although the defects observed appear consistent with inhibition of Hedgehog signaling, we do not know why a “full spectrum” of Shh-associated defects are not observed if perturbation of Shh signaling is responsible for the defects. However, difference in tissue response could be linked to differential distribution of PBO in the embryo and the relative sensitivity of each developmental system to Shh perturbation (i.e., how much of an effect on Shh signaling is necessary to alter development in each system). Overall, these reports suggest that adverse effects due to inhibition of Hedgehog signaling may be particularly deleterious during early development. Current EPA documents state a low degree of concern for fetal susceptibility to the effects of PBO (Daiss, 2006). Our data suggests that additional studies are needed.

PBO has other toxicities that may be unrelated to its Hedgehog inhibitory effect. The major target organ of induced toxicity is the liver (Daiss, 2010). PBO induces hepatocarcinogenesis in rats fed a diet containing either 1.2 or 2.4% PBO for 2 years (Takahashi et al., 1994). Mechanistic studies suggest that PBO can generate reactive oxygen species and induce hepatocellular tumors (Muguruma et al., 2007, 2009) with the addition of 0.25% PBO in the diet reported as a threshold dose that induces reactive oxygen species–mediated hepatocarcinogenesis in rats (Kawai et al., 2010; Muguruma et al., 2009).

Our unexpected discovery that PBO is a Smo inhibitor highlights the importance of using target-based, especially developmental pathway-dependent, screening strategies to unveil previously unknown toxicities of environmental compounds. Utilizing target-based studies to expose underlying biology is particularly important in cases where concerns have been raised as a result of clinical observations or epidemiological studies, such as recently occurred for PBO (Horton et al., 2011).

The proliferation of chemicals like PBO into the environment has prompted regulatory agencies to actively seek new technology for generating risk assessments for public health and policy decisions. Our work strongly supports policy initiatives aimed at using innovative strategies for toxicity testing, such as Tox21 (Schmidt, 2009). Our findings address the unmet challenges for toxicity testing and risk assessment of environmentally ubiquitous chemical agents and demonstrate that the daunting task involved in evaluating large numbers of chemical substances can be overcome.

Overall, our study demonstrates that PBO is a Smo antagonist that inhibits the Hedgehog signaling pathway, a critical regulator of stem cell proliferation, organ development and homeostasis, cancer, and central nervous system development. Considering (1) the widespread presence of PBO in the environment, (2) the recent epidemiologic association of PBO exposure with delayed mental development in children, and (3) our findings that PBO inhibits the Hedgehog signaling pathway, the safety profile of PBO needs to be investigated further. Our findings also demonstrate that innovative high-throughput drug screening technology can be highly valuable in evaluating large numbers of environmental toxicants and identifying key targets and unknown biological activity helpful in generating chemical/biological risk assessments for public health and environmental policy decisions.

SUPPLEMENTARY DATA

Supplementary data are available online at http://
toxsci.oxfordjournals.org/.

FUNDING

National Institutes of Health (5R01CA113656-03 to W.C.); Fred and Alice Stanback (H.K.L.); the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health (Z01ES102045 to J.H.F).

ACKNOWLEDGMENTS

We thank Robert J. Lefkowitz, Margaret Kirby, Richard Premont, Gregory Michelotti, and Edward Levin for helpful discussions. W.C. is a V Foundation Scholar and an American Cancer Society Scholar.

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