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. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: Mol Cancer Ther. 2016 Feb 12;15(5):911–921. doi: 10.1158/1535-7163.MCT-15-0521

Pre-Clinical Evaluation of UAB30 in Pediatric Renal and Hepatic Malignancies

Alicia M Waters 1, Jerry E Stewart 1, Venkatram R Atigadda 2, Elizabeth Mroczek-Musulman 3, Donald D Muccio 2, Clinton J Grubbs 1, Elizabeth A Beierle 1
PMCID: PMC4873350  NIHMSID: NIHMS760715  PMID: 26873726

Abstract

Rare tumors of solid organs remain some of the most difficult pediatric cancers to cure. These difficult tumors include rare pediatric renal malignancies such as malignant rhabdoid kidney tumors (MRKT) and non-osseous renal Ewing sarcoma, and hepatoblastoma, a pediatric liver tumor that arises from immature liver cells. There are data in adult renal and hepatic malignancies demonstrating the efficacy of retinoid therapy. The investigation of retinoic acid therapy in cancer is not a new strategy, but the widespread adoption of this therapy has been hindered by toxicities. Our laboratory has been investigating a novel synthetic rexinoid, UAB30, which exhibits a more favorable side effect profile. In this current study, we hypothesized that UAB30 would diminish the growth of tumor cells from both rare renal and liver tumors in vitro and in vivo. We successfully demonstrated decreased cellular proliferation, invasion and migration, cell cycle arrest and increased apoptosis after treatment with UAB30. Additionally, in in vivo murine models of human hepatoblastoma or rare human renal tumors, there were significantly decreased tumor xenograft growth and increased animal survival after UAB30 treatment. UAB30 should further be investigated as a developing therapeutic in these rare and difficult to treat, pediatric solid organ tumors.

Keywords: malignant rhabdoid kidney tumor, hepatoblastoma, retinoid, 9-cis-UAB30, HuH6, SK-NEP-1, G401

Introduction

Rare tumors of solid organs remain some of the most difficult pediatric cancers to cure. These difficult tumors include pediatric renal malignancies such as malignant rhabdoid kidney tumors (MRKT) and non-osseous renal Ewing sarcoma. MRKTs make up 2% of pediatric kidney tumors [1] but unfortunately, the overall ten year survival is less than 30% [2]. Renal Ewing sarcoma represents another aggressive renal tumor with 25–50% of children presenting with advanced disease [3, 4]. Hepatoblastoma, a pediatric liver tumor that arises from immature liver cells, like these rare renal malignancies, also provides a significant therapeutic challenge to the pediatric cancer caregiver, having a dismal survival rate of only 50% [5]. Novel and innovative therapeutic interventions are needed for the treatment of these aggressive solid organ malignancies, especially high risk, non-responsive and relapsing tumors.

Retinoid therapy is currently the standard of care for maintenance treatment in other pediatric malignancies including acute promyelocytic leukemia and the solid tumor, neuroblastoma [6]. A novel retinoid, 9-cis-UAB30 (UAB30), a synthetic analog of 9-cis-retinoic acid, binds selectively to the retinoid X receptor (RXR) leading to activation of genes involved in induction of differentiation and apoptosis [7, 8]. UAB30 has minimal toxicity compared to other retinoids [7, 9 – 11], and has recently been found to be effective in decreasing xenograft tumor growth in neuroblastoma [12]. Investigators have demonstrated the effectiveness of retinoids against adult liver [13] and kidney tumors [14]. These data in neuroblastoma [12], and the prior results with retinoids in adult renal and hepatic malignancies [13, 14], led us to believe that UAB30 may have an effect upon tumorigenicity in rare pediatric renal and hepatic malignancies.

We hypothesized that the treatment of the rare pediatric renal tumors, MRKT and renal Ewing sarcoma, and hepatoblastoma with UAB30 would impact tumor growth in vitro and in vivo. We demonstrated cell cycle arrest, decreased proliferation and motility, and apoptosis in vitro and decreased xenograft growth in vivo with UAB30 treatment. These studies suggested a potential role for UAB30 in the treatment of aggressive pediatric solid organ tumors.

Methods

Cells and Cell Culture

Cell lines were maintained at 37 °C and 5% CO2. Human hepatoblastoma cell line, HuH6, was from Thomas Pietschmann (Hannover, Germany) [15] and maintained in Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum (FBS) (Hyclone, Suwanee, GA), 1 μg/mL penicillin/streptomycin (Gibco, Carlsbad, CA), and 2 mM L-glutamine (Thermo Fisher Scientific, Rockford, IL). The MRKT cell line, G401 (CRL-1441), was from American Type Culture Collection (ATCC, Manassas, VA) [16] and maintained in McCoy’s medium containing 10% non-heat-inactivated FBS (Hyclone), 1 μg/mL penicillin/streptomycin (Gibco), and 2 mM L-glutamine (Fisher). The renal Ewing sarcoma cell line, SK-NEP-1 (ATCC, HTB-48) [17], was maintained in McCoy’s medium (30-2007, ATCC) containing 15% FBS (Hyclone), 1 μg/mL penicillin/streptomycin (Gibco) and 2 mM L-glutamine (Fisher). Due to non-adherent nature, SK-NEP-1 cells were passed by centrifugation at 300 rpm for 5 minutes, aspiration of the supernate, and resuspension of the pelleted live cells in medium. All cell lines were obtained within last 4 years, were mycoplasma free, but were not characterized.

Antibodies and Reagents

Rabbit polyclonal anti-PARP (9542S), anti-caspase 3 (9662), anti-AKT (9272), anti-phospho AKT (S473, 9271), anti-ERK1/2 (9102), and mouse monoclonal anti-β-actin antibodies were from Cell Signaling Technology (Danvers, MA). Rabbit anti-phospho ERK1/2 (05-797R), mouse anti-FAK (4.47) and anti-phospho Src (Y416, 05-677) was from Millipore (EMD Millipore, Billerica, MA). Mouse monoclonal anti-RXR was from Abcam (clone MOK13-17, Cambridge, MA), rabbit anti-Src from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-phospho FAK (Y397) from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA). 9-cis-UAB30 (UAB30) was synthesized as described [18].

Antibodies for immunofluorescence: primary anti-RXR antibody (Abcam, 1:1000) and secondary goat anti-mouse Alexa Fluor 594 antibody (A-11045, Thermo Fisher, 1:33 dilution).

Immunoblotting

Western blots were performed as described [19]. Whole cell lysates were isolated using radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors (Sigma Aldrich, St. Louis, MO), phosphatase inhibitors (Sigma) and phenylmethanesulfonylfluoride. Lysates were centrifuged at 14 000 rpm for 30 min at 4 °C, protein concentrations determined with BCA Protein Assay Reagent (Pierce, Rockford, IL) and separated by electrophoresis on sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels. Antibodies were used according to manufacturer’s recommendations. Molecular weight markers (Precision Plus Protein Kaleidoscope Standards, Bio-Rad, Hercules, CA) confirmed the expected size of the target proteins. Immunoblots were developed with Luminata Classico or Crescendo ECL (EMD Millipore). Blots were stripped with stripping solution (Bio-Rad) at 37 °C for 15 minutes and then re-probed with selected antibodies. Equal protein loading was confirmed with β-actin.

Cell Viability/Proliferation Assays

Cell viability was measured with alamarBlue® assays. Cells (1.5 × 103 per well) were plated on 96-well culture plates, allowed to attach, and treated with UAB30 at increasing concentrations for 48 hours. Following treatment, 10 μL of alamarBlue® dye (Invitrogen) was added and absorbance (595 nm) was measured using a microplate reader (BioTek Gen5, BioTek Instruments, Winooski, VT). Viability was reported as fold change. Cell proliferation was measured with CellTiter96® AQueous One Solution (Promega, Madison, WI) and trypan blue staining. Cells (5 × 103 cells per well) were plated, allowed to attach and treated with UAB30 for 48 hours. Following treatment, 10μL CellTiter96® AQueous One Solution (Promega) was added and the absorbance at 490 nm was measured using a kinetic microplate reader (BioTek). For trypan blue, cells (1.5 × 103 cells per well) were plated, treated, stained with trypan blue and counted with a hemacytometer and reported as fold change in cell count and fold change in the ratio of dead to viable cells.

Apoptosis

Multiple methods were used to detect apoptosis. Cells were treated with increasing concentrations of UAB30 and whole cell lysates immunoblotted for cleavage of poly ADP-ribose polymerase (PARP). Detection of PARP cleavage indicated apoptosis. In addition, apoptosis was shown with a caspase 3 activation kit (KHZ0022, Invitrogen). Briefly, cells (1.5 × 106) were plated and treated with increasing concentrations of UAB30 for 48 hours, lysed and protein determination assays completed to assure equal protein loading. Reagents were added to the proteins and the colorimetric changes were detected using a microplate reader (405 nm, BioTek) and results reported as fold change in caspase 3. Caspase 3 was also detected with immunoblotting. Decreased total protein or increased cleavage products indicated apoptosis.

Cell Cycle Analysis

Cells (1.0 × 106) were allowed to attach and then treated with UAB30 (10 μM, 48 hours). Cells were trypsinized, washed with phosphate buffered saline (PBS), and fixed in 100% ethanol. Ethanol was removed and cells stained with propidium iodide (PI) solution containing 0.3 μM PI (Invitrogen) in 0.1% Triton X and RNAse A (Qiagen, Valencia, CA) for 30 minutes at room temperature (RT), and analyzed with fluorescence-activated cell sorting (FACS) using a FACSCalibur™ Flow Cytometer (Becton Dickinson Biosciences, San Jose, CA). Data were analyzed with ModFit LT software (Verity Sofware House Inc., Topsham, ME). Negative controls were included in each FACS run.

Cellular Invasion Assay

Twelve-well culture plates (TransWell®, Corning Inc., Lowell, MA) with 8 μm micropore inserts were coated with Matrigel™ (BD Biosciences) (2 mg/mL, 50 μL for 4 hours at 37 °C) on the top side. Cells (G401, HuH6) were treated with UAB30, plated into the upper well (4 × 104 cells), and allowed to invade for 48 hours. The inserts were fixed with 3% paraformaldehyde, stained with crystal violet, cells counted with a light microscope and invasion reported as fold change.

Attachment Independent Growth Assay

Attachment independent growth was determined by soft agar assay. A base layer of culture media in 1% noble agar was established and SK-NEP-1, G401 and HuH6 cells (1×104) were plated in the top layer of culture media and agar. Dishes were treated with UAB30 (0, 10 μM) every 4 days. After for 4 – 6 weeks, colonies were imaged and quantified using Gel Dock Imager (Bio-Rad) and Quantity One Software (Bio-Rad) and colony counts reported as fold change.

Migration Assays

Similar to invasion, culture plates (TransWell®, Corning) with 8 μm micropore inserts were used with collagen Type I (10 mg/mL, 50 μL for 4 hours at 37 °C) coating the bottom side of the insert. Cells (G401, HuH6) (4 × 104) were treated with UAB30, placed into the upper well, and allowed to migrate for 24 hours. The inserts were then fixed with 3% paraformaldehyde, stained with crystal violet, and migrated cells counted with a light microscope. Migration was reported as fold change.

Cellular migration was also measured utilizing a cell monolayer wounding (scratch) assay. G401 and HuH6 cells (3.5 × 105) were plated and allowed to attach. A 200 μL pipette tip created a uniform scratch in the cell layer and photos [Photometrics CoolSNAP HQ2 CCD camera (Tucson, AZ) attached to a Nikon Eclipse Ti microscope (Tokyo, Japan)] were obtained at time zero. Cells were treated with UAB30 (0- 20 μM) for 24 hours and photos repeated. The area of the scratch was quantified by comparing the pixel count of the scratched area to the pixel count of the same plate at time zero and reported as fold change in closure. Invasion, migration and wounding assays were not performed utilizing SK-NEP-1 cells since they do not propagate in an adherent fashion and floating cells do not lend well to these assays [20].

Immunofluorescence

Immunofluorescence staining was utilized to detect movement of RXR into the nucleus following UAB30 (10 μM). Cells were plated on glass chamber slides, allowed to attach and treated with UAB30. The SK-NEP-1 cell line did not grow well on these slides because they propagate as floating cells and were therefore not analyzed. After 48 hours, cells were fixed with 3% paraformaldehyde, permeabilized with 0.15% Triton X-100, and primary antibody added at RT for 1 hour. The Alexa Fluor 594 secondary antibody was added (RT, 45 minutes). Prolong Gold with DAPI (P36931, Invitrogen) was used for mounting. Imaging was performed with a Zeiss LSM 710 Confocal Scanning Microscope with Zen 2008 software (Carl Zeiss MicroImaging, LLC, Thornwood, NY) using a 63× objective with a zoom of 0.9. MetaMorph® Microscopy Image Analysis Software (Ver. 7.6, Analytical Technologies, Molecular Devices, Sunnyvale, CA) analyzed the images.

In Vivo Tumor Growth

Six week old, female, athymic nude mice (Harlan Laboratories, Inc., Chicago, IL) were maintained in the specific pathogen free facility with standard 12 hour light/dark cycles with chow and water ad libitum. Experiments were approval by the Institutional Animal Care and Use Committee (140209355) and conducted within institutional, national and NIH guidelines. Human MRKT cells, G401 [2.5 × 106 cells in 25% Matrigel™ (BD Biosciences)], or human hepatoblastoma cells, HuH6 [2.5 × 106 cells in 25% Matrigel™ (BD Biosciences)], were injected into the right flank. On the day of injection, mice were randomized to receive daily administration of vehicle-treated or UAB30-treated (100 mg/kg body weight/day) chow (n = 10 per group). Previous experiments proved this dosage to be well tolerated [21]. Tumor volumes were measured with calipers and calculated with the standard formula (width2 × length)/2 where length is the largest measurement. Animals were followed until IACUC parameters for euthanasia were met (6 or 7 weeks, HuH6 and G401, respectively), at which point all animals were euthanized with CO2 and bilateral thoracotomy and the tumors harvested.

Human renal Ewing sarcoma cells, SK-NEP-1 (1.5 × 106 cells/50 μL sterile PBS) were injected into the sub-capsular space of the left kidney in six week old, female, athymic nude mice (Harlan Laboratories). When injected into the orthotopic position these cells will form lung metastasis [20, 22]. At injection, animals were randomized to daily administration of vehicle-treated or UAB30-treated (100 mg/kg body weight/day) chow (n = 10 per group). After four weeks of treatment, the animals were euthanized and the kidney tumors and lungs harvested.

Detection of Metastases

Three levels from each lung of the animals with SK-NEP-1 xenografts were formalin fixed (10% buffered formalin), paraffin embedded and stained with hematoxylin and eosin. These specimens were examined by a board certified pediatric pathologist (EMM) to determine the presence or absence of metastases.

Immunohistochemistry

Formalin-fixed, paraffin-embedded human specimens and xenograft tumor specimens were cut (6 μM sections), baked at 70 °C for 1 hour, deparaffinized, rehydrated and steamed. Sections were quenched with 3% hydrogen peroxide and blocked with blocking buffer (BSA, powdered milk, Triton X-100, PBS) for 30 minutes at 4 °C. The primary RXR (ab2815, Abcam) or anti-Ki67 antibody (ab15580, Abcam) was added (1:200) and incubated overnight at 4 °C. Secondary antibody (1:400, rabbit anti-mouse SuperPicture Polymer HRP, Invitrogen, or donkey anti-rabbit, Jackson ImmunoResearch Laboratories, respectively) was added for 1 hour at 22 °C. Staining was developed with VECTASTAIN Elite ABC kit (PK-6100, Vector Laboratories, Burlingame, CA), TSA™ (biotin tyramide reagent, 1:400, PerkinElmer, Inc., Waltham, MA) and 3,3′-diaminobenzidine (DAB) (ImmPACT DAB, Vector Laboratories, Burlingame, CA). Slides were counterstained with hematoxylin. Negative controls [mouse IgG (1 μg/mL, Invitrogen) or rabbit IgG (1 μg/mL, EMD Millipore)] were included with each run.

A board-certified pathologist (EMM) blinded to the treatment groups completed the Ki67 quantification. Area chosen for analysis was that of greatest immunoreactivity. Five hundred cells were counted and the ratio of immunopositive to total cells reported as percent positive staining [23, 24].

Data Analysis

Experiments were repeated at least in triplicate, and data reported as mean ± standard error of the mean. Student’s t-test, Fisher’s exact test, Chi square, or ANOVA was used as appropriate with statistical significance determined at p<0.05.

Results

RXR expression

To determine whether UAB30, an RXR agonist, would have a receptor target, the expression of RXR receptors was evaluated. Immunoblotting detected RXR expression in all 3 cell lines (Fig. 1A). In addition, UAB30 treatment led to increased percentage of RXR staining in the nucleus in the G401 and HuH6 cell lines (p ≤ 0.03) (Fig. 1B, Supplemental Data Figure 1), indicating that UAB30 functioned as an RXR agonist. RXR staining in human renal tumors and hepatoblastoma specimens was examined. Human specimens were obtained from the tumor repository at our institution (IRB: X10093009; X110825022). Positive staining was seen in 8 of 8 hepatoblastoma specimens and in both of the MRKT and renal Ewing tumors examined. Representative photomicrographs at 40 × are presented (Fig. 1C). Negative controls were included with each run (inserts left corners).

Figure 1. RXR expression.

Figure 1

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A G401, SK-NEP-1 and HuH6 whole cell lysates were examined with immunoblotting for the RXR receptor. RXR was seen in all 3 cell lines. B Immunofluorescence staining and confocal microscopy was used to detect RXR staining in the nucleus with and without treatment of UAB30 (10 μM, 48 hrs). The staining of RXR in the nucleus was calculated with Metamorph and reported as percent nuclear staining. UAB30 resulted in increased nuclear staining of RXR in G401 and HuH6 cell lines. C Immunohistochemistry for RXR was performed on renal and hepatoblastoma formalin-fixed, paraffin-embedded human specimens. RXR staining (brown) was seen in both the MRKT and Ewing renal tumors and in hepatoblastoma samples. Representative photomicrographs at 40× with insert showing negative controls (left corner). Immunofluorescence data represents three independent experiments.

UAB30 led to cell death

Next, the effects of UAB30 treatment upon cell survival were evaluated with alamarBlue® assays. UAB30 treatment resulted in significant cell death in the renal tumors and hepatoblastoma cell lines (Fig. 2A and B, p ≤ 0.05, respectively). The LD50 for UAB30 was calculated: G401, 30 ± 4 μM; SK-NEP-1, 22 ± 2 μM; and HuH6, 78 ± 9 μM. To determine whether UAB30-induced cell death involved apoptosis, immunoblotting was performed for PARP cleavage. As demonstrated by increased PARP cleavage (Fig. 2C), apoptosis was present. Reduction of total caspase 3 was detected by immunoblotting in all three cell lines at 100 μM UAB30 (Supplemental Data Figure 2) and activation of caspase 3 was significantly increased in all three cell lines (p ≤ 0.05) by treatment with UAB30 (50 μM) (Fig. 2D), further demonstrating apoptosis.

Figure 2. UAB30 decreased cell survival and apoptosis.

Figure 2

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A G401 and SK-NEP-1 cell lines were treated with UAB30 at increasing concentrations. After 48 hours, cell viability was measured with alamarBlue™ assays. Data were reported as mean ± standard error of the mean. There was a significant decrease in viability in both cell lines following UAB30 treatment beginning at 10 μM. B HuH6 cell line was treated with UAB30 at increasing concentrations. After 48 hours of treatment, cell viability was measured with alamarBlue™ assays. Data reported as mean ± standard error of the mean. There was a significant decrease in viability following UAB30 treatment starting at 20 μM. C The effects of UAB30 on apoptosis. SK-NEP-1, G401 and HuH6 cells were treated with UAB30 at increasing concentrations for 48 hrs and lysates examined with immunoblotting for PARP cleavage products. There was an increase in cleaved PARP at the 50 μM concentration in all cell lines. D Apoptosis was also detected with caspase 3 activation. Cells were treated with UAB30 for 48 hrs prior to study. Cleaved caspase 3 was increased in the G401 cell line at 40 μM UAB30, and in the other two cell lines at 50 μM, indicating apoptosis. Survival data represent 4 independent experiments and apoptosis via caspase 3 cleavage is represented by 3 independent experiments.

UAB30 resulted in cell cycle arrest

Since retinoids are known to arrest cell cycle [25], we wished to determine whether UAB30 would have similar effects in these cell lines. Concentrations of UAB30 (10 μM) were chosen that were below the calculated LD50 to demonstrate early morphologic changes rather than cell death. Representative FACS histograms are presented both pre- (control, left panels) and post-UAB30 (right panels) treatment (Fig. 3A). UAB30 led to arrest in cell cycle G0/G1 progression in all cell lines with an increased percentage of cells in G1 (p ≤ 0.05) and decreased percentage of cells in S phase (p ≤ 0.05) (Fig. 3B).

Figure 3. UAB30 induced cell cycle arrest and decreased proliferation.

Figure 3

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Cells were treated with 10 μM UAB30 for 48 hrs and cell cycle was analyzed with propidium iodide staining and flow cytometry. A Representative FACS histograms of cells before (left panel) and after (right panel) treatment with UAB30, showed an increase in the percentage of cells in the G1 phase and a decrease in the S phase. B FACS cell cycle data in graphic form; black bars represent percentage of cells in G1, dark grey represent the percentage of cells in G2 and light grey represents the percentage of cells in S phase. In all cell lines UAB30 significantly increased percentage of cells in G1 and decreased percentage in S phase. C Cellular proliferation was measured with CellTiter96®. Cells were treated with UAB30 for 48 hours. There was a significant decrease in proliferation in all three cell lines after treatment. Cell cycle and proliferations assays were repeated for three independent experiments.

After finding cell cycle arrest, we wished to determine if UAB30 would also affect cellular proliferation. CellTiter96® and trypan blue exclusion was performed to measure proliferation. In all three cell lines proliferation decreased following UAB30 treatment (10 μM, 48 hours) (p ≤ 0.05) (Fig. 3C). In the trypan blue assays, there was a decrease in total cell number (Supplemental Data Figure 3A) and the ratio between dead cells and viable cells significantly increased (p ≤ 0.05) (Supplemental Data Figure 3B). Together, these data indicated that UAB30 diminished cellular proliferation.

UAB30 decreased cell invasion and migration

Next we wished to determine if UAB30 would alter other phenotypic features of the tumor cells. The hallmark of aggressive tumor cells is their ability to invade and migrate. To study the effects of UAB30 upon cellular invasion and migration, G401 and HuH6 cells were treated with low concentrations of UAB30 (up to 10 μM) for 48 hours. There was a significant decrease in cellular invasion at 5 μM concentration of UAB30 in both cell lines that was even more enhanced at 10 μM (p ≤ 0.01) (Fig. 4A).

Figure 4. UAB30 led to decreased cell invasion and migration.

Figure 4

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A G401 and HuH6 cells were plated in TransWell® culture plates with Matrigel™ coating the top side of the insert. Cells were treated with UAB30, allowed to invade for 48 hrs, and then fixed, stained and counted, with invasion reported as fold change. There was a significant decrease in invasion in both cell lines beginning at 10 μM UAB30. B Invasion was also measured with anchorage independent growth. SK-NEP-1, G401 and HuH6 cells were plated at 1× 104 cells in a media and agar mixture. Dishes were treated with UAB30 (0, 10 μM) and retreated every 4 days. After 4 – 6 weeks, colonies were imaged and quantified, and colony counts reported as fold change. There was a significant decrease in colony formation in all cell lines following treatment with UAB30. C G401 and HuH6 cells were plated in TransWell® culture plates with the insert coated with collagen Type 1. Cell lines were treated with increasing concentrations of UAB30 and allowed to migrate for 24 hrs. The cells were fixed, stained and counted and migration reported as fold change. Migration was significantly inhibited in both the G401 and the HuH6 cell lines at 5 μM concentration of UAB30. Migration was further examined with monolayer wounding assays. G401 and HuH6 cells were plated, allowed to attach and a standardized scratch was performed. Cells were treated with increasing concentrations of UAB30 and photographs of the plates were obtained after 24 hrs. D Representative photographs of the G401 (upper panel) and the HuH6 (lower panel) migration inserts. UAB30 treatment decreased the number of cells migrating through the membrane. There was a For the HuH6 cell lines, dashed lines represent distance of original scratch, solid lines the distance remaining after 24 hrs. Invasion and migration data represent 5 independent experiments and the attachment independent growth represents 4 independent experiments.

Attachment independent growth (soft agar assays) is considered one of the best measures of cellular invasion, and was used to measure invasion in the G401, SK-NEP-1 and HuH6 cell lines. Cells were treated with UAB30 (10 μM), placed into soft agar, and colonies were allowed to grow for 4–6 weeks. The number of colonies detected at the end of the studies was decreased by 34% (p ≤ 0.01), 26% (p ≤ 0.05) and 86% (p ≤ 0.01) in the UAB30-treated G401, SK-NEP-1, and HuH6 cell lines, respectively, compared to controls (Fig. 4B).

The migration of tumor cells was studied in a fashion similar to invasion. Again, concentrations of UAB30 used to treat TransWell® plates (0–10 μM) were significantly less than the LD50. Treated cells were allowed to migrate for 24 hours. There was a significant decrease in the number of migrating cells beginning at 5 μM UAB30 when compared to controls (p ≤ 0.01) (Fig. 4C). Representative photographs of the stained inserts demonstrate decreased migration following UAB30 treatment (Fig. 4D). Migration was also studied with a monolayer scratch assay. The UAB30-treated cells showed a significant decrease in their migration across a monolayer scratch injury (Supplemental Data Figure 4A, B). The SK-NEP-1 cell line was not studied with TransWell® invasion or migration or scratch assay as these cells propagated in both an adherent and non-adherent fashion, which limited the evaluation of this cell line by these methods.

UAB30 decreased tumor growth in vivo in a nude mouse model of pediatric renal tumors

To more easily monitor tumor growth, a flank xenograft model was used to study the effects of UAB30 upon malignant rhabdoid kidney tumor growth. G401 cells (2.5 × 106), were injected into the right flank of female athymic nude mice (n = 20). On the day of injection, animals were randomized to either standard chow (control, vehicle) or chow with UAB30 added (n = 10/group). UAB30 was administered at a dose (100 mg/kg body weight/day) previously shown to be well tolerated by this species [21]. Tumors were measured for 7 weeks. The tumors in the G401 control-treated animals grew more rapidly and were significantly larger at seven weeks than the UAB30 treated animals (662 ± 189 mm3 vs. 36 ± 13 mm3, control vs. UAB30, p = 0.003) (Fig. 5A). Xenograft tumors were stained for Ki67 to determine proliferation. Ki67 decreased in the UAB30 treated tumors, but differences were not statistically significant. This finding was likely secondary to the fact that the control tumors were large and necrotic, leaving fewer cells that could proliferate, compared to the UAB30 tumors that although small, had more viable cells and therefore, more cells capable of proliferation.

Figure 5. UAB30 treatment resulted in decreased tumor growth in a nude mouse model of renal tumors.

Figure 5

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A G401 cells (2.5 × 106), were injected into the right flank of female athymic nude mice (n = 20) and animals randomized to receive standard chow (control, vehicle) or chow with UAB30 (100 mg/kg body weight/day) (n = 10/group). Tumors were measured for 7 weeks. Tumors in control-treated animals grew more rapidly and were significantly larger at euthanasia than the UAB30 treated animals (660 ± 189 mm3 vs. 36 ± 13 mm3, control vs. UAB30, p = 0.003). B An orthotopic nude mouse model was used to evaluate UAB30 on the growth and metastasis of SK-NEP-1, renal Ewing sarcoma cells. SK-NEP-1 cells (1.5 × 106) were injected into the subcapsular space of the left kidney of female athymic nude mice (n = 28), and the animals were randomized to standard chow (control, vehicle, n = 13) or UAB30 chow (100 mg/kg body weight/day) (n = 15). After four weeks, all animals were euthanized and kidney tumors harvested. C Animals treated with UAB30 had significantly smaller tumor weights (0.6 ± 0.1 g vs. 1.5 ± 0.2 g, UAB30 vs. control, p = 0.003) and D volumes (204 ± 50 mm3 vs. 630 ± 134 mm3, UAB30 vs. control, p = 0.007) compared to controls. E Representative photomicrograph (40×) demonstrating a pulmonary metastasis (black arrow) of SK-NEP-1 tumor in the lung of a control treated animal.

An orthotopic nude mouse model was used to evaluate UAB30 upon the growth of renal Ewing sarcoma cells and their ability to form lung metastasis [20, 22]. SK-NEP-1 cells (1.5 × 106) were injected into the subcapsular space of the left kidney of female athymic nude mice (n = 28). On the day of injection, mice were randomized to standard chow (control, vehicle, n = 13) or chow with UAB30 (100 mg/kg body weight/day) (n = 15). After four weeks, all animals were euthanized and kidney tumors harvested (Fig. 5B). Previously, we showed that four weeks were required for control animals to reach pre-moribund tumor growth [20]. In addition, the un-injected kidney (right) was also weighed to serve as a control. Animals treated with UAB30 had significantly smaller tumor weights (0.6 ± 0.1 g vs. 1.5 ± 0.2 g, UAB30 vs. control, p = 0.003) (Fig. 5C) and volumes (204 ± 50 mm3 vs. 630 ± 134 mm3, UAB30 vs. control, p = 0.007) (Fig. 5D) than controls. Animals that received UAB30 demonstrated less weight gain than the control animals, therefore, a tumor weight to animal weight ratio was calculated to determine if the lack of weight gain could explain the differences in tumor weight. The UAB30 treated animals had a significantly smaller ratio than the control animals (Supplemental Data Figure 5), which demonstrated that the treatment, rather than the smaller animal size, accounted for the decreased kidney tumor growth. In addition, there was no difference between the weights of the control, contralateral right kidney, between the two groups, also providing evidence that the lack of weight gain was not responsible for the significant difference in tumor growth. Ki67 staining was completed on these xenografts tumors, and again tended to be higher in the untreated tumors, but did not reach statistical significance. Since SK-NEP-1 tumors are known to metastasize to the lungs [20, 22], lung tissues were stained with H & E to detect metastases. A representative example of a pulmonary metastasis (40×) in a control treated animal is presented in Figure 5E. The samples were evaluated by a board certified pathologist (EMM) blinded to the treatment groups. Any sized metastasis was counted as a positive. Lung metastases were significantly more common in control animals (54% vs. 13%, p ≤ 0.05, control vs. UAB30).

UAB30 decreased tumor growth in a nude mouse model of hepatoblastoma

An in vivo model of hepatoblastoma tumor growth following UAB30 treatment was employed using female athymic nude mice. HuH6 hepatoblastoma cells (2.5 × 106 in Matrigel™) were injected into the right flank of each mouse (n = 20). On the day of injection, mice were randomized to standard chow (control, vehicle) or chow with UAB30 (100 mg/kg body weight/day) (n = 10/group). Tumors were measured for 6 weeks, at which time all the control tumors reached IACUC parameters for euthanasia, and all animals were euthanized. The tumors in the HuH6 control-treated animals were significantly larger at six weeks than those from the UAB30 treated animals (2440 ± 366 mm3 vs. 657 ± 175 mm3, control vs. UAB30, p = 0.0002) (Fig. 6A). These tumor xenografts were stained for Ki67 to measure cellular proliferation. Representative photomicrographs of Ki67 staining are presented (Fig. 6B). There was a decrease in cell proliferation in the UAB30 treated animals compared to controls, but it did not reach statistical significance (80% vs. 58%, control vs. UAB30, p = 0.1). Again, the control tumors were large and mostly necrotic, leaving fewer viable cells available for proliferation than the much smaller UAB30 treated tumors, likely leading to these findings.

Figure 6. UAB30 decreased tumor growth in a nude mouse model of hepatoblastoma.

Figure 6

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A HuH6 hepatoblastoma cells (2.5 × 106 in Matrigel™) were injected into the right flank of female athymic nude mice (n = 20), and animals randomized to receive standard chow (control, vehicle) or UAB30 chow (100 mg/kg body weight/day) (n = 10/group) for 6 weeks. The tumors in the HuH6 control-treated animals were significantly larger than those from the UAB30 treated animals (2440 ± 366 mm3 vs. 657 ± 175 mm3, control vs. UAB30, p = 0.0002). B Tumor xenografts were stained for Ki67 to measure cellular proliferation. Representative photomicrographs of Ki67 staining are presented (40×). Ki67 staining is represented by brown color and inserts (left lower corner) show staining for negative control.

Discussion

The investigation of retinoids for the treatment of cancer has been ongoing for over 25 years, but other than in neuroblastoma [26], it has not become widely used for pediatric solid tumors. In recent literature, retinoids have been shown to have some efficacy for adult renal cell carcinoma (RCC) prolonging both median time to disease progression and overall survival [27, 28]. In hepatocellular carcinoma (HCC), retinoids are currently being studied for chemoprevention [29–31]. These studies prompted the current investigation of UAB30 in these difficult to treat pediatric solid tumors involving the kidney and liver.

In this study we demonstrated that the nuclear receptor, RXR, was present in human hepatoblastoma specimens and in the HuH6 hepatoblastoma cell line. To our knowledge, this finding is novel. The RXR receptors have been documented in HCC cell lines [32–34], a primarily adult hepatic malignancy, so it was not completely unexpected that this nuclear receptor would be present in hepatoblastoma, and abnormalities in the RXR receptors have been highly associated with HCC progression [35, 36]. There were not significant differences in the RXR staining in the human hepatoblastoma specimens and therefore staining could not be correlated with patient survival or disease progression. However, demonstration of RXR expression was important to providing evidence that this therapy was clinically reasonable for hepatoblastoma.

Likewise, RXR receptor protein was found in MRKT and non-osseous renal Ewing sarcoma specimens and cell lines, also not previously documented. RXR receptors have been studied in RCC, a predominantly adult renal malignancy. Buentig et al found that nuclear location of RXR inferred a survival advantage [37] implying that functional RXR receptors decreased RCC tumorigenicity. Further, Obara showed that the loss of RXR expression was associated with advanced RCC [38]. Similar to the hepatoblastoma specimens, the renal tumors specimens had fairly uniform staining, but the lack of a large number of specimens for review prevented a comparison between stain score and patient outcomes. Like hepatoblastoma, the presence of RXR in these specimens did lend credence to the potential for use of an RXR agonist in these tumors.

Multiple kinases have been hypothesized to be involved in retinoic acid effects upon cellular differentiation and apoptosis. Zhang et al showed that all-trans-retinoic acid (ATRA) combined with cisplatin reduced AKT phosphorylation of HCC cells [39]. ERK1/2 activation has also been shown to be induced by fenretinide in HepG2 (HCC) cells [39], but these same investigators showed that fenretinide decreased phosphorylation of ERK 1/2 in other HCC cell lines [40]. Others showed that ATRA treatment of HCC cells resulted in increased JNK phosphorylation [41]. These findings highlight the conflicting data that exist regarding retinoids and downstream pathways. Extensive investigations of retinoid-induced kinase activation have not been completed in renal tumor cell lines, but data from other tumor types have indicated that focal adhesion kinase (FAK) or Src may be involved. Our studies found no changes in phosphorylation in a number of these kinases previously shown to be relevant to retinoid treatment of hepatic or renal tumors (Supplemental Data Figure 6). Our data did indicate that the therapeutic activity of UAB30 may be related to increasing the RXR activity in the nucleus (Fig. 1B). Other investigators have demonstrated that increases in retinoid receptor mRNA abundance or proteins correlated with retinoid-induced growth inhibition in RCC [42] and HCC [34, 43]. Therefore, the mechanisms are likely more related to restoration of retinoid receptors responsible for controlling the expression of differentiation and other retinoic acid-responsive genes than to changes in kinase signaling.

The number of cell lines available for study of these rare, deadly, pediatric solid tumors is limited. One of the cell lines that we chose was the SK-NEP-1 cell line. This cell line was originally thought to be Wilms tumor, but has since been characterized as a renal Ewing sarcoma [17]. SK-NEP-1 was chosen since renal Ewing sarcomas, similar to MRKTs, are clinically much more difficult to treat and carry a significantly worse prognosis than the standard Wilms tumor [44].

The dose of UAB30 chosen for the in vivo treatments was based upon our previous experience. The animals were given UAB30 a dose of 400 mg/kg diet which translated to 100 mg/kg body weight/day [45]. The animals tolerated this dose without significant changes in mucous membranes or skin. The animals with the orthotopic kidney tumor implant did not gain weight as quickly as their control counterparts; but their smaller size did not result in a difference in tumor: body weight ratios (Supplemental Data Figure 5) indicating that the differences in body growth were not responsible for the decreased tumor size. In the adult trials that have been conducted, the patients given UAB30 did not experience changes in appetite or weight loss. Although the adults did not lose weight, unlike children, they were not required to gain weight and grow during their exposure to UAB30. The decreased growth in the animals will raise the question as to the effects of UAB30 upon growth in children and will require investigation as this treatment moves forward into the clinical arena.

Currently, retinoic acid is utilized in pediatric cancer treatment algorithms in the course of treatment other than established disease [26]. Therefore, we chose to incorporate this same model in our animal studies, looking at the ability of UAB30 to prevent tumor establishment or metastasis. The findings that UAB30 is a better differentiating agent than cytotoxic agent, similar to other retinoids, also made this model a more attractive one for the current studies. Future studies combining UAB30 with cytotoxic agents will obviously require utilization of an established disease model.

In the current study we demonstrated that a novel rexinoid, UAB30, was able to decrease cell survival in both kidney and hepatic tumor cell lines. In addition, UAB30 led to changes in cellular phenotypes that resulted in cell cycle arrest and decreased migration and invasion in vitro. There was also significantly decreased tumor growth in flank xenograft and orthotopic murine tumor models. These results suggested that UAB30 may have a therapeutic role in these difficult to treat pediatric solid malignancies.

Supplementary Material

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Acknowledgments

Financial Support: This work was funded by the UAB Comprehensive Cancer Center Mortimer A. and Josephine B. Cohen Research Acceleration and Innovation Fund (EA Beierle) and in part by institutional grants from the National Cancer Institute including T32CA091078 (AM Waters), flow cytometry core grants P30 AR048311 (UAB) and P30 AI027767 (UAB), and high resolution imaging core grants P30 CA013148 (UAB) and P30 AR048311 (UAB).

Special thanks to Shawn Williams and the UAB High Resolution Imaging Core for confocal microscopy assistance.

Abbreviations

13-cis RA

13-cis-retinoic acid

ANOVA

analysis of variance

FACS

fluorescence-activated cell sorting

mRNA

messenger ribonucleic acid

EMEM

Eagle’s minimal essential medium

HCC

hepatocellular carcinoma

LD50

lethal dose, 50%

RIPA

radioimmunoprecipitation assay

PARP

Poly ADP-ribose polymerase

PBS

phosphate buffered saline

RCC

renal cell carcinoma

SDS-PAGE

sodium dodecyl sulfate polyacrylamide

SPF

specific pathogen free

Footnotes

Conflict of Interest: D.D. Muccio and V.R. Atigadda hold a US Patent for the methods of making UAB30 and the uses thereof. Patent number 11/661,030, August 23, 2005. None of the other authors have any conflicts of interest.

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Supplementary Materials

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NIHMS760715-supplement-1.docx (18.1KB, docx)
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NIHMS760715-supplement-2.tif (2.9MB, tif)
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NIHMS760715-supplement-3.tif (508.3KB, tif)
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NIHMS760715-supplement-4.tif (294.4KB, tif)
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NIHMS760715-supplement-5.tif (2.1MB, tif)
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