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Genomics

CRISPR Knockout of the HuR Gene Causes a Xenograft Lethal Phenotype

Shruti Lal, Edwin C. Cheung, Mahsa Zarei, Ranjan Preet, Saswati N. Chand, Nicole C. Mambelli-Lisboa, Carmella Romeo, Matthew C. Stout, Eric Londin, Austin Goetz, Cinthya Y. Lowder, Avinoam Nevler, Charles J. Yeo, Paul M. Campbell, Jordan M. Winter, Dan A. Dixon and Jonathan R. Brody
DOI: 10.1158/1541-7786.MCR-16-0361 Published June 2017

Abstract

Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the United States, whereas colorectal cancer is the third most common cancer. The RNA-binding protein HuR (ELAVL1) supports a pro-oncogenic network in gastrointestinal (GI) cancer cells through enhanced HuR expression. Using a publically available database, HuR expression levels were determined to be increased in primary PDA and colorectal cancer tumor cohorts as compared with normal pancreas and colon tissues, respectively. CRISPR/Cas9 technology was successfully used to delete the HuR gene in both PDA (MIA PaCa-2 and Hs 766T) and colorectal cancer (HCT116) cell lines. HuR deficiency has a mild phenotype, in vitro, as HuR-deficient MIA PaCa-2 (MIA.HuR-KO(−/−)) cells had increased apoptosis when compared with isogenic wild-type (MIA.HuR-WT(+/+)) cells. Using this isogenic system, mRNAs were identified that specifically bound to HuR and were required for transforming a two-dimensional culture into three dimensional (i.e., organoids). Importantly, HuR-deficient MIA PaCa-2 and Hs 766T cells were unable to engraft tumors in vivo compared with control HuR-proficient cells, demonstrating a unique xenograft lethal phenotype. Although not as a dramatic phenotype, CRISPR knockout HuR HCT116 colon cancer cells (HCT.HuR-KO(−/−)) showed significantly reduced in vivo tumor growth compared with controls (HCT.HuR-WT(+/+)). Finally, HuR deletion affects KRAS activity and controls a subset of pro-oncogenic genes.

Implications: The work reported here supports the notion that targeting HuR is a promising therapeutic strategy to treat GI malignancies. Mol Cancer Res; 15(6); 696–707. ©2017 AACR.

Introduction

Pancreatic ductal adenocarcinoma (PDA), with an overall 5-year survival rate of roughly 7 percent, is one of the deadliest solid tumors (1, 2). Colorectal cancer is the third most common cancer in the United States and the fourth most common cause of death worldwide (3). Both colorectal cancer and PDA tumorigenesis are associated with numerous sequential genetic changes (4). However, methods to target common mutations (KRAS, p16/DSKN2A, TP53) in PDA and colorectal cancer have been particularly challenging and shown limited therapeutic value (5, 6). Genetic mutations and copy-number changes are critical regulatory mechanisms promoting the initiation and progression of tumorigenesis. Beyond driving mutations, it is becoming clearer that posttranscriptional gene regulatory mechanisms play an important role in controlling the available transcriptome (7). Posttranscriptional gene regulation characteristically involves trans-acting miRNAs and RNA-binding proteins (RBP) that are potent regulators controlling the abundance of specific pro-oncogenic proteins (8, 9).

Hu proteins are a family of trans-acting RBPs that recognize and bind to cis-acting AU-rich RNA elements (ARE) within the mRNA 3′ untranslated regions (10). The RBP HuR (ELAVL1) is expressed ubiquitously and is extensively involved in mRNA stability and translation. HuR targets, which include proto-oncogenes, cytokines, growth, and invasion factors, implicate an important role of HuR in cancer development (11). HuR is abundant in both PDA and colorectal cancer specimens compared with normal adjacent tissue with cytoplasmic subcellular localization associated with increased tumor stage (12, 13). As tumor cells are under stress in the tumor microenvironment (ref. 14; e.g., nutrient deprivation and hypoxia; refs. 15, 16), HuR translocates from the nucleus and binds to pro-oncogenic mRNA transcripts (e.g., WEE1, PIM1, COX-2, VEGF), promoting cancer cell survival and tumorigenesis (14, 17, 18).

To further depict the role of HuR in GI cancer biology, we genetically deleted HuR in PDA and colorectal cancer cells by using clustered, regularly interspaced, short palindromic repeat (CRISPR)/Cas9 technology. The system efficiently targets a specific genomic sequence and then creates disruptive double-strand breaks (DSB). The DSB is repaired via nonhomologous end joining and thus insertions/deletions (INDEL) are randomly placed at the sites of repair (19). These INDELs can lead to deleterious frameshift mutations and ultimately premature stop codons to effectively disrupt the targeted gene on one (+/–) or both (–/–) alleles, and thus allowing an efficient way to generate an isogenic cell culture model in which to study the importance of HuR in GI cancer cells.

Materials and Methods

Evaluation of HuR mRNA in pancreas clinical specimens

Previously reported microarray expression (20) data were downloaded from GEO (accession number: GSE71729). This dataset contains 46 normal pancreatic tissue samples, 145 primary pancreatic adenocarcinomas, 42 normal colon, and 286 primary colon adenocarcinomas. The relative expression level for HuR was extracted from the downloaded matrix and used for comparisons between the three groups of samples.

Cell culture, transfections, and treatments

Cell culture conditions.

MIA PaCa-2, Hs 766T, and HCT116 cells were purchased from ATCC. Cells were cultured in DMEM supplemented with 10% FBS (Gibco/Invitrogen), 1% l-glutamine (Gibco/Invitrogen), and 1% penicillin–streptomycin (Invitrogen) at 37°C in 5% humidified CO2 incubators.

Transfections.

MIA PaCa-2 and Hs 766T cells were seeded in 6-well plate at the density of 500,000 cells per well for 24 hours. Cells transfected with approximately 5 μg of CRISPR control and HuR knockout plasmids using Lipofectamine 2000 (Gibco/Invitrogen) as previously described (14). Cells were harvested after 48 hours of transfection, and GFP-positive cells were single sorted into 96-well plates using FACS Calibur flow cytometer (BD Biosciences).

MIA.HuR-KO(–/–) cells were seeded in 6-well plate at the density of 500,000 cells per well, and cells were transfected with HuR overexpression plasmid as previously described (21).

Treatments.

Cells were treated with the IC50 values of the DNA-damaging agent MMC (mitomycin C; Sigma), gemcitabine (Eli Lilly), and oxaliplatin (Sigma) for 24 hours as previously described (14) by adding directly into the culture medium.

All experiments using PDA CRISPR-transfected cells were performed on cells passage between 10 to 25.

Plasmid design

CRISPR knockout in MIA PaCa-2 and Hs 766T PDA cell lines.

Three different guide RNAs of HuR fused with CRISPR-cas9 and GFP protein were designed and purchased from Sigma (Supplementary Fig. S1A and Supplementary Table S1). Guide RNA 1 was located on exon 2 (sense), guide RNA 2 was also located on exon 2 (antisense), and guide RNA 3 was located on exon 3 (sense; Supplementary Table S1). The CRISPR Universal Negative Control plasmid was purchased from Sigma (Cat. No. CRISPR06-1EA). Plasmids were purified using a Qiagen's plasmid mini-purification kit following the manufacturer's protocol (Qiagen).

In the figures, the MIA.HuR-WT(+/+) clone is “MIA.HuR,” MIA.HuR-KO(+/+) clone is “Clone.4,” MIA.HuR-KO(−/−).1 clone is “Clone.11,” MIA.HuR-KO(−/−).2 clone is “Clone.14,” and MIA.HuR-KO(−/−) + HOE is “Clone.11 stably transfected with an HuR overexpression plasmid.”

CRISPR knockout in HCT116 colon cancer cell line.

CRISPR/Cas9-mediated knockout of ELAVL1 in the human colon cancer cell line HCT116 (ATCC) was accomplished using HuR-CRISPR/Cas9 KO plasmid (sc-400141; Santa Cruz Biotechnology), followed by homology-directed repair (HDR) insertion of a puromycin resistance gene/red fluorescent protein (RFP) cassette (sc-400141-HDR; Santa Cruz Biotechnology) into the DSB according to the vendor's protocol. Transfected cells were selected in normal growth medium containing 0.5 μg/mL puromycin (Thermo Fisher Scientific) for 2 to 3 weeks. Individual clones were isolated using cloning cylinders, and HuR knockout clones screened by Western blotting for HuR and genotyped by PCR to verify exon insertion of the Puro/RFP cassette. For stable cell maintenance, the puromycin concentration was reduced to 0.2 μg/mL.

Sequencing analysis

Sanger sequencing was performed using sequencing primers listed in Supplementary Table S2 on genomic DNA of the clones. The Sequencher software (Gene Codes Corporation) was used to analyze the chromatogram and identify mutation sites. Sequence changes were classified according to the HGVS nomenclature recommendation and based on the NCBI reference sequence: NM_001419.2. Clone sequencing and expression data are provided in Supplementary Table S4 and Supplementary Fig. S1B and S1C. For simplicity, the clones employed in our experiments will be referred to as MIA.HuR-KO(−/−).1 (Clone.11, created with Guide RNA 3), MIA.HuR-KO(−/−).2 (Clone.14, created with Guide RNA 3), MIA.HuR-KO(+/+) (Clone.4, created with Guide RNA 1), and HsT.HuR-KO(−/−) (created with Guide RNA 3). These clones correlate with NM_001419[c.211delCG];[c.211delCG], NM_001419[c.204delGTGACCGCGA];[c.206delGACCGCGAAGGATGCAG], NM_001419 wild type, and NM_ 001419[c.211_212insATGATAGTCCATTTTAAAACATAATTTTAAAACTGCAAACTAC];[c.211_212insATGATAGTCCATTTTAAAACATAATTTTAAAACTGCAAACTAC] mutation sites, respectively.

Whole-cell extracts and SDS-PAGE/Western blotting

Whole-cell lysates were isolated using RIPA lysis buffer (Invitrogen) by incubating the cell pellets on ice for 10 minutes followed by centrifugation at 13,000 × g for 15 minutes at 4°C as previously described (14). Samples were then mixed 4:1 with 5X Laemmli buffer and boiled for 5 minutes. Proteins were measured with the Pierce BCA Kit (Thermo Fisher Scientific), and approximately 50 μg was separated using a 12% Bis-Tris polyacrylamide gel and transferred to PVDF membrane (Invitrogen), blocked in 1:1 Licor Odyssey blocking buffer, and incubated overnight at 4°C with α-Tubulin (Santa Cruz Biotechnology; #sc-5286) and HuR (Santa Cruz Biotechnology; #sc-5261) antibodies. Protein complexes were then visualized with Licor Odyssey.

Immunofluorescence

Approximately 50,000 cells were plated on coverslips per well in 24-well plate. After 24 hours, cells were treated with MMC, gemcitabine, and oxaliplatin for 24 hours. Cells were then washed 2 times with 1X PBS, fixed with 4% paraformaldehyde for 10 minutes at room temperature, permeabilized with 0.25% Triton-X for 30 minutes, blocked with 5% goat serum for 1 hour, and incubated overnight with HuR antibody, as previously described (14). DAPI was used to stain cell nuclei, and coverslips were mounted with DAPI ProLong Gold Antifade (Invitrogen). Slides were visualized with a Zeiss LSM-510 Confocal Laser Microscope. All images were taken at 40X magnification with oil. Images were cropped using AIM browser.

Flow cytometry analysis

Cleaved caspase 3.

Cells were seeded at 300,000 cells per well in 6-well plates for 24 hours. Apoptosis was measured by incubating the cells for 30 minutes with the CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (ThermoFisher Scientific), according to the manufacturer's instructions, and analyzed using a FACS Calibur flow cytometer (BD Biosciences). Calculated relative mean fluorescence intensity was produced using FlowJo software (Tree Star, Inc.).

Cell cycle.

Cells were seeded at 500,000 cells per well in 6-well plates for 24 hours. Cells were pulse chased with bromodeoxyuridine (BrdU; Amersham) by adding directly to actively growing cells for 1 hour as previously described (14) and analyzed on FACS Calibur flow cytometer. Graphs were generated on Excel (Microsoft).

Soft-agar assay

A bottom layer of 0.75% agar was prepared in complete medium in 60 mm tissue culture dishes. A top layer of 0.36% agar supplemented with 10,000 cells/mL was added as previously described (14). Pictures were taken using Floid Cell Imaging Station (Life Technologies), and colonies were counted using ImageJ (http://imagej.nih.gov/ij/).

Organoid cultures

Cell lines were obtained from two-dimensional culture (standard cell culture) and washed with cold organoid basic media. Cells are then spun at 1,200 rpm for 5 minutes and washed again. The pellet was suspended in Matrigel, and 50 μL of cell–Matrigel mixture is added to each well of a prewarmed 24-well plate. The plate was then placed in a standard tissue culture incubator until the Matrigel solidified. Note that 500 μL of prewarmed complete organoid feeding media is then added to each well. The plates are placed in a tissue culture incubator and inspected on a daily basis as described by Boj and colleagues and Baker and colleagues (22, 23). For all organoid cultures and studies, we validated HuR knockout for the CRISPR lines via qRT-PCR analysis (data not shown).

Image analysis was performed using Paint Shop Pro (Version 7.04, Corel Corporation) and R (Version 3.3.2, the R Foundation for statistical computing). Microscopic images of identical magnification were digitally acquired at three time points (1280 × 960 Pixels). Images were centered and cropped to 800 × 800 pixels. Organoid objects maps of the images were created using gray scale value thresholding and also visually assessed and corrected for merging and segmentation errors. The produced maps were assessed for size and number of objects per map using the EBImage: Image processing and analysis toolbox for R (http://rdrr.io/bioc/EBImage/). Objects with surface area smaller than 10 pixels were excluded from the analysis. Cell line organoid sizes were assessed for normality of distribution with the Kolmogorov–Smirnov test (24). Area size was compared using the Mann–Whitney test for nonparametric distributions and t test for normal distributions (25). P values < 0.05 were considered as statistically significant.

Serum starvation assay

MIA.HuR-WT(+/+) and MIA.HuR-KO(−/−) cells growing in log phase in normal growth medium (RPMI 1640 + 10% FBS) were serum starved (0.1% FBS) for 16 or 72 hours. Cells were homogenized in lysis buffer [25 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol, 10 mmol/L MgCl2, 1x phosphatase, and protease inhibitors (HALT, Thermo)], and protein concentration was quantified by BCA assay (Pierce). A Ras-GTP pulldown was performed according to previously established protocols (26, 27). Western blot analysis was performed on the pulldown and straight protein lysate samples blotting for K-Ras and K-Ras-GTP (Calbiochem; #OP24), AKT (Cell Signaling Technology; #4691), pAKT (Cell Signaling Technology; #4060), MEK (Cell Signaling Technology; #8727), pMEK (Cell Signaling Technology; #9154), and Vinculin (Sigma; #V9131). Protein bands were visualized by Luminata ECL reagents (Millipore) on x-ray film or a FluorChem M (Protein Simple) imager. Protein bands were analyzed by densitometry and normalized for protein load against vinculin and relative to MIA.HuR-WT(+/+) signals.

MTT assay

Cells were seeded at 5,000 cells/well in 96-well tissue culture plates. Cell growth was assayed using the MTT-based cell growth determination Kit (Sigma-Aldrich) as previously described (28). Cell growth was calculated as relative absorbance (A570) normalized to cells after 1 day of growth. Data are represented as average of three independent experiments ± SEM.

Short-term drug sensitivity assay

Cells were seeded at 1,000 cells/well in 96-well tissue culture plates in triplicates. Cells were treated 24 hours later and exposed to only one dose. After 7 days of treatment, cells were washed, lysed, stained with PicoGreen (Invitrogen), and fluorescence intensity was read as previously described (29). Graph was generated using GraphPad. Data are represented as average of three independent experiments ± SEM.

RNP-IP and qPCR

Cells were plated at 50% confluency in 100 mm dishes. Cells were either left untreated or treated with 1 μmol/L oxaliplatin for 24 hours. Immunoprecipitation was performed using either anti-HuR or IgG control antibodies as previously described (14). Total RNA was isolated, and WEE1, IDH1, and PARP1 mRNA binding was validated by RT-qPCR.

In vivo experiment

MIA PaCa-2 and Hs 766T xenografts.

The experiments involving mice received the approval of the Thomas Jefferson University Institutional Animal Care regulations and Use Committee (IACUC). Six-week-old, female, athymic nude mice were purchased from Harlan Laboratories, and 5 × 106 cells were injected subcutaneously in the left and right flanks, respectively (5 mice per group). Cells were prepared in 100 μL solution comprised of 70% DPBS and 30% Matrigel. Tumor volumes and body weight were measured 3 times per week using a caliper, and tumor volumes were calculated using the formula Volume = Length × Width2/2. Upon termination of the experiment, mice were euthanized using carbon dioxide inhalation followed by cervical dislocation, and tumors were harvested.

HCT116 xenografts.

Athymic nude (Nu/Nu) mice were purchased from The Jackson Laboratory and maintained under sterile conditions in cage micro-isolators according to approved IACUC guidelines. Parental HCT116 and HuR knockout clones 1 and 2 (2 × 106 cells) used between passages 14 and 23 were resuspended in PBS containing 50% Matrigel (Corning) and injected into the dorsal subcutaneous tissue. Tumor growth was assayed as described (28, 30).

Results

HuR mRNA is more abundant in primary GI tumors compared with normal tissue

We have previously demonstrated that HuR is upregulated in colorectal cancer cells (13). To determine HuR expression levels in pancreatic samples, we analyzed previously reported microarray data (20) of HuR mRNA levels in normal pancreatic tissues (n = 46) and primary tumors (n = 145) as well as in colon normal tissues (n = 42) and primary tumors (n = 286). We detected that HuR mRNA levels are enhanced slightly in both pancreatic (P < 0.005) and colon (P < 0.0005) primary tumors compared with normal tissues (Fig. 1A).

Figure 1.
Figure 1.

CRISPR-cas9 system inhibits HuR expression and localization in MIA PaCa-2 PDA cells. A, Shown are boxplots of HuR expression levels across 519 samples comprised of normal pancreatic tissue (n = 46), primary pancreatic adenocarcinomas (n = 145), normal colon tissue (n = 42), and colorectal cancer (n = 286). For each boxplot, the black line represents the median expression value, and the boxes are the first and third quartiles. **, P < 0.005 and ***, P < –0.005. B, Chromatograms from Sanger sequencing of HuR-CRISPR–transfected purified PCR products showing the homozygous mutant [MIA.HuR-KO(−/−).1] and homozygous wild-type [MIA.HuR-KO(+/+)] clones. C, Relative mRNA HuR expression was assessed in different clones by qPCR. (Note: only three clones are shown here. Complete list of clones is shown in Supplementary Fig. S1B.) D, Relative protein expression of HuR was assessed in different clones by immunoblot. (Note: this is a photoshop cropped image of three clones shown in Supplementary Fig. S1C.) E, HuR nuclear and cytoplasmic translocation was assessed by immunofluorescence upon MMC, gemcitabine (GEM), and oxaliplatin (Oxa) stressors. MIA.HuR-WT(+/+) in B to E is a product from MIA PaCa-2 cells with CRISPR control transfection. WT, wild type and HM, homozygous mutant. Note: In B–E, the MIA.HuR-WT(+/+) clone is “MIA.HuR.”

Successful generation of HuR knockout PDA cells using the CRISPR-Cas9 system

We transfected the PDA cell line MIA PaCa-2 cells, known to harbor common genetic lesions found in most PDAs (K-RAS, p53, and p16; ref. 31), with three different guide RNAs designed to target exons 2 and 3 of human HuR (ELAVL1; Supplementary Table S1). A plasmid containing Cas9 protein fused with GFP and guide RNA expressed from a single vector was transfected to MIA PaCa-2 cells (Supplementary Fig. S1A). As a control, a nonspecific guide RNA in this vector was also transfected. Forty-eight hours after transfection, cells were single sorted with FACS into 96-well plates. The mutation was confirmed by Sanger sequencing in expanded single-cell colonies (Supplementary Tables S2, S3, and S4; Fig. 1B). Only guide RNA 3 targeting the exon 3 of HuR resulted in producing 5 stable clones (Fig. 1C and D; Supplementary Fig. S1B and S1C; Supplementary Tables S3 and S4). HuR mRNA levels were analyzed by qPCR (Fig. 1C; Supplementary Fig. S1B), and protein expression was detected by immunoblotting (Fig. 1D; Supplementary Fig. S1C) to demonstrate HuR gene expression correlated to the genotype. Both MIA.HuR-WT(+/+) (wild-type parental CRISPR control transfected cells showing homozygous wild-type genotype) and MIA.HuR-KO(+/+) (HuR-CRISPR–transfected cells showing homozygous wild-type genotype; Supplementary Tables S3 and S4) clones displayed similar levels of HuR nucleo-cytoplasmic expression upon MMC, gemcitabine, and oxaliplatin stressors by immunofluorescence (Fig. 1E), indicating functional HuR translocation to the cytoplasm from the nucleus upon specific stress (14). The MIA.HuR-KO(−/−) (HuR-CRISPR–transfected cells showing homozygous knockout/mutant genotype; Supplementary Tables S3 and S4) cells showed no sign of HuR expression (Fig. 1E). We also created a CRISPR HuR knockout in another PDA cell line Hs 766T using guide RNA 3. The mutation in HuR was again confirmed by Sanger sequencing (Supplementary Fig. S1D; Supplementary Table S4), and functional inactivation of HuR was confirmed by immunoblot analysis (Supplementary Fig. S1E). These data demonstrate that the HuR gene was successfully knocked out using CRISPR-Cas9 system in multiple PDA cell lines.

HuR-deficient PDA cells have a mild phenotype in vitro

To determine the effect of HuR deletion has on the cancer phenotype, HuR knockout clone MIA.HuR-KO(−/−).1 along with controls, MIA.HuR-WT(+/+) and MIA.HuR-KO(+/+), were analyzed for in vitro growth viability with a trypan blue exclusion assay. Results demonstrated that MIA.HuR-KO(−/−).1 induced higher amounts of cell death compared with controls (Fig. 2A). In addition, cleaved caspase 3 activity, a marker of cell death, was measured with FACS, and similar results were obtained (Fig. 2B and C), indicating HuR's role in inhibiting apoptotic cell death. Taken together, these data demonstrate that the complete loss of HuR induces more cell death consistent with our previous work (32).

Figure 2.
Figure 2.

HuR-deficient cells induce more cell death. A, Cells were stained with Trypan blue, and number of dead cells was counted after 24, 48, and 72 hours for one clone depicting each mutation. Graphs are average of three individual experiments. *, P < 0.003 and **, P < 0.004. B and C, Cell death was also assessed by cleaved caspase 3 activity after 24 hours of cell seeding via FACS. Graphs are average of three individual experiments. *, P < 0.0004. D and E, Long-term cell survival assay was assessed by colony formation soft-agar assay. Graphs are average of two individual experiments. *, P < 0.003. F, Cell kinetics was measured by pulse chasing the cells by BrdU followed by FACS. Graphs are average of two individual experiments.

We have performed drug sensitivity assay (short-term cell survival) using a chemotherapeutic drug MMC and under glucose deprivation and observed that MIA.HuR-KO(−/−) cells are sensitive compared with controls (Supplementary Fig. S2A and S2B). To determine the long-term cell survival and anchorage independence growth, soft-agar assays were performed. Results showed that loss of HuR (in the MIA.HuR-KO(−/−).1 model) resulted in approximately 5-fold decrease in colony formation compared with controls (Fig. 2D and E). Lastly, the effects of HuR on cell cycle were analyzed by pulse-labeling the cells with BrdU. Shown in Fig. 2F, MIA.HuR-KO(−/−).1 cells are primarily arrested in S phase with reduced number of cells in the G2–M phase, indicating that cells were cycling through mitosis and most likely prematurely dying.

In an effort to study a malignant characteristic of the HuR-deficient cells and how they would establish under the stressful transition from moving into a three-dimensional (3D) culture, we evaluated the ability of each cell line's capacity to transform into organoids (22). After 7 to 10 days of plating, MIA.HuR-KO(+/+) cells formed spheroids easily in 3D cultures within in a passage, whereas MIA.HuR-KO(−/−).2 cells only formed single-cell sheets and were unable to form substantial spheroids even after 4 weeks of plating (Supplementary Fig. S2C and S2D). Image analysis of the spheroid structures in each group revealed the MIA.HuR-KO(+/+) spheroids to be significantly larger than the MIA.HuR-KO(−/−).2 spheroids (5–10- fold increase in cross-section area, P < 0.01, Supplementary Fig. S2D). Furthermore, during the course of 2 weeks, the MIA.HuR-KO(+/+) continued to grow and doubled in size (P < 0.01) compared with the MIA.HuR-KO(−/−).2 spheroids. Similarly, when HsT.HuR-WT(+/+) and HsT.HuR-KO(−/−) cells were plated in 3D cultures, the HsT.HuR-WT(+/+) demonstrated more robust spheroid formation and growth than the HsT.HuR-KO(−/−) cells (data not shown). These data suggest that HuR is critical for initiation of growth in a stressful transition to 3D and also viability in a more relevant in vitro 3D culture (22).

Using isogenic MIA.HuR-WT(+/+) and MIA.HuR-KO(−/−) PDA cells to identify HuR targets

It was previously published that HuR translocates to the cytoplasm upon specific stress where it binds to its mRNA targets and stabilizes their transcript and/or increases the protein translation (33). We and others have demonstrated that Ribonucleotide immunoprecipitation (RNP-IP) assays (34) are a reliable and efficient way to capture HuR-bound mRNA targets (14, 17, 32, 33, 35). To evaluate binding of HuR to target mRNAs, two MIA.HuR-KO(−/−) (MIA.HuR-KO(−/−).1 and MIA.HuR-KO(−/−).2) cell lines along with controls MIA.HuR-WT(+/+) and MIA.HuR-KO(+/+) cells were treated with oxaliplatin (1 μmol/L) for 24 hours to induce cytoplasmic translocation (Fig. 3A). Cytoplasmic lysates were subjected to HuR immunoprecipitation (Fig. 3B), followed by analysis of HuR targets IDH1 and WEE1 by qPCR. Shown in Fig. 3C, PDA cells deficient in HuR showed background levels of IDH1 and WEE1 mRNA, whereas control cells showed increased binding of HuR to these targets upon oxaliplatin stress. PARP1, a nontarget of HuR, showed no significant binding in knockout as well as in controls samples (Fig. 3C).

Figure 3.
Figure 3.

HuR-deficient cells failed to bind to its targets. A and B, RNP-IP assay was performed to determine the binding of HuR to its mRNA targets. Cells were treated with oxaliplatin (1 μmol/L) for 24 hours and immunoprecipitated using either control IgG or HuR antibodies. Western blot showing HuR expression in cytoplasmic and nuclear lysates and immunoprecipitated lysates. C, HuR binding to WEE1 and IDH1 mRNA was analyzed by qPCR. PARP1 is a nontarget of HuR.

HuR is required for PDA tumor growth

Because we were able to obtain viable HuR-deficient cancer cell lines (see Fig. 1B–E) and the in vitro phenotype was not dramatic for the MIA PaCa-2 line (Fig. 2), we evaluated if loss of HuR would disrupt cancer cell viability and growth in vivo. To test the effect of HuR knockout on in vivo PDA tumor growth, we first performed two individual experiments utilizing nude mice subcutaneously injected in their hind flanks with equal numbers of MIA.HuR-WT(+/+), MIA.HuR-KO(+/+), and two of different MIA.HuR-KO(−/−) cells (MIA.HuR-KO(−/−).1 and MIA.HuR-KO(−/−).2). Tumor volumes and body weight were measured 3 times per week and harvested after 48 days of tumor growth. Shown in Fig. 4A, tumors derived from the MIA.HuR-WT(+/+) and MIA.HuR-KO(+/+) groups were 1,976.7 mm3 and 1,305.8 mm3, respectively. The mean tumor volumes generated from MIA.HuR-KO(+/+) cells were not significantly smaller than those from MIA.HuR-WT(+/+) cells (P < 0.716; Fig. 4B and P < 0.589; Supplementary Fig. S3A). However, this is in sharp contrast to both MIA.HuR-KO(−/−).1 and MIA.HuR-KO(−/−).2 groups, where tumor growth was not observed (Fig. 4A and B) and reproducible in an independent experiment (Supplementary Fig. S3A and S3B). To then validate these results, we performed the same experiment in Hs 766T HuR knockout cell line. Nude mice subcutaneously injected with HsT.HuR-WT(+/+) and HsT.HuR-KO(−/−) cells showed similar growth results as in MIA PaCa-2 cell line (Figs. 4C and D).

Figure 4.
Figure 4.

HuR knockout by CRISPR inhibits the PDA xenograft growth. A, Representative images of subcutaneous tumors on the flanks of nude female mice and representative pictures of corresponding excised tumors (day 48). B, Tumor growth curves of MIA.HuR-WT(+/+), MIA.HuR-KO(+/+), MIA.HuR-KO(−/−).1, and MIA.HuR-KO(−/−).2 xenografts in nude mice shown in A. Data represent mean ± SEM of n = 5. ns, non-significant; *, P < 0.01; and **, P < 0.003. C, Representative images of subcutaneous tumors on the flanks of nude female mice and their corresponding excised tumors injected with CRISPR HuR (HsT.HuR-KO(−/−)) and CRISPR Control (HsT.HuR-WT(+/+)) cells in Hs 766T cell line (day 33). D, Tumor growth curves in HsT.HuR-WT(+/+) and HsT.HuR-KO(−/−) xenografts in nude mice shown in C. Data represent mean ± SEM of n = 4. ns, non-significant and *, P < 0.0003. E, Representative images of subcutaneous tumors on the flanks of nude female mice and representative pictures of corresponding excised tumors (day 48). F, Tumor growth curves of MIA.HuR-WT(+/+), MIA.HuR-KO(−/−).1, and MIA.HuR-KO(−/−).1+ HOE xenografts in nude mice shown in E. Data represent mean ± SEM of n = 5. ns, non-significant; *, P < 0.005; and **, P < 0.006.

Adding HuR in a HuR-null background rescues the tumor growth phenotype

To validate that loss of HuR was responsible for the observed lack of tumor growth, we created a stable cell line by overexpressing HuR in MIA.HuR-KO(−/−).1 cells and assaying xenograft tumor growth. Results demonstrated that xenografts with MIA.HuR-KO(−/−).1 cells overexpressing HuR [labeled as (MIA.HuR-KO(−/−))+ HOE] restored the tumor formation (1,061.4 mm3 vs. 0 mm3; P < 0.0002), demonstrating that HuR plays a vital role in PDA growth (Fig. 4E and F). These findings using a PDA xenograft model demonstrate that targeted genetic disruption of HuR may prove to be a novel therapeutic strategy to inhibit tumor growth as a treatment for PDA.

HuR knockout suppresses xenograft growth in a colon cancer model

In order to determine the effect of HuR knockout on cancer models other than PDA, we created HuR knockouts in the colorectal carcinoma HCT116 cell line. Previously, we have demonstrated that HuR is upregulated in HCT116 and other colorectal cancer cell lines (13, 18). A similar CRISPR/Cas9-mediated approach was taken, except that a puromycin resistance gene/RFP cassette was inserted into cleavage sites within ELAVL1 exons 3, 4, or 5 by HDR. Clones were screened for puromycin resistance and RFP expression and then selected loss of HuR expression (Fig. 5A). Confirmation of cassette insertion by PCR-based genotyping identified 2 HuR knockout clones (HCT.HuR-KO(−/−).1 and HCT.HuR-KO(−/−).2). Similar to our PDA model, short-term in vitro growth assays of HCT.HuR-KO(−/−) cells displayed attenuated growth compared with the control (Fig. 5B). To determine the effects of HuR loss in colon cancer xenografts, we subcutaneously injected nude mice in their hind flanks with equal number of HCT.HuR-WT(+/+) and HCT.HuR-KO(−/−) cells. Tumor volumes were measured on alternate days and harvested after 35 days. Results showed that tumors in the HCT.HuR-KO(−/−) group were dramatically smaller than control HCT.HuR-WT(+/+) (Fig. 5C, D, and E), consistent with what was observed with PDA cells. Similar results were also observed in HCA7 colon cancer cells with CRISPR/Cas9-mediated deletion of HuR (data not shown). These results demonstrate that targeted genetic disruption of HuR inhibits cancer growth in both PDA and colorectal cancer models.

Figure 5.
Figure 5.

CRISPR/Cas9 knockout of HuR in colon cancer cells retards cell and tumor growth. A, Western blot analysis of HuR expression in parental HCT116 cells (labeled HCT.HuR-WT(+/+)) and HuR knockout clones (HCT.HuR-KO(−/−).1 and HCT.HuR-KO(−/−).2). Total protein extracts were analyzed by SDS-PAGE and probed for HuR; actin served as a loading control. B, In vitro cell growth was measured by MTT assay. Relative absorbance (A570) was normalized to cells after 1 day of growth. Cells were used between passages 5 to 12. Data are represented as average of three independent experiments ± SEM. C, In vivo tumor growth of HCT116 cells and HuR knockout clones 1 and 2 (passages 23, 14, and 16, respectively) xenografts in nude mice. Tumor volume (mm3) is represented as average of seven tumors ± SEM. D and E, Average tumor weight ± SEM and representative tumors excised at day 35 are shown. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

HuR-deficient PDA cells have decreased K-Ras activation

Because disrupted K-Ras signaling is a hallmark of GI cancers and previously deletion of K-ras caused a similar dramatic in vivo phenotype (36), we sought to define the role HuR has on K-Ras activation. Under normal growth conditions, a loss of HuR results in an increase in K-Ras and AKT proteins, but interestingly, not a concomitant significance increase in their respective activations (Fig. 6A and B). Under acute (16 hours) serum deprivation, MIA.HuR-WT(+/+) cells respond by increasing K-Ras activation, whereas HuR knockout cells can no longer mount this stress response and show a significant decline in GTP loading of K-Ras.

Figure 6.
Figure 6.

Loss of HuR diminishes stress-induced K-Ras activation. Immunoblot depiction of total and activated (GTP-bound) K-Ras and AKT protein in MIA PaCa-2 cells lacking HuR under 16- (A) or 72-hour (C) serum deprivation, respectively. Quantitative analysis of immunoblots for acute or (B) or 72 hours (D), * P < 0.05 HuR(−/−) compared with MIA.HuR.WT(+/+), n = 3 to 6 independent experiments. Protein bands are normalized to vinculin and are relative to MIA.HuR.WT(+/+) signals.

Upon extended (72 hours; Fig. 6C and D) serum deprivation, MIA.HuR-KO(−/−).1 cells no longer maintained the high levels of compensatory K-Ras expression seen in the acute serum starvation, and again K-Ras activation was blunted (unlike HuR-competent cells, where K-Ras-GTP was increased approximately 6- ± 2-fold in 72 hours starved vs. normal growth conditions). Although MIA.HuR-KO(−/−).1 cells were capable of augmenting AKT expression and activation in normal growth conditions, this survival pathway activation was diminished under chronic serum starvation. These data suggest that in PDA, survival mechanisms that are in part mitigated by oncogenic, activated KRas can be regulated by HuR function under stressed conditions. Thus, proteins responsible for proper KRas activation (i.e., posttranslational modification, trafficking, GEF/GAP activity ratio, etc.) may be targets of HuR binding and mRNA stabilization.

Discussion

CRISPR/Cas9-mediated HuR knockout is a novel way to investigate HuR biology because previous studies relied on RNAi-based approaches using shRNA-inducible knockdowns or siRNA transient transfections (33), where leakiness in the constructs and incomplete knockdown can lead to inconsistent results. If amenable, a genomic deletion is an effective and unbiased way to evaluate gene function at a more fundamental level. Furthermore, this approach allows for identifying off-target effects of molecularly targeted therapies where the target is not present in the cell-based model. Using this approach, we deleted HuR in MIA PaCa-2, Hs 766T, and HCT116 cells, and demonstrated an ablation of HuR protein expression compared with wild-type and CRISPR control cells. These commonly used cell models of GI cancer will advance the field for studying HuR biology in PDA and colorectal cancer, along with facilitating current drug discovery where HuR is a target in preclinical models (14, 17, 18, 21, 32, 33, 35, 37–39). However, it should be noted that at the time of acceptance, we found that the later passaged MIA.HuR-KO(−/−).1 line seemed to also contain a population having an HuR (wild-type) allele (as determined by Sanger sequencing). We have re-validated the HuR depletion at early passages where experiments herein were performed (data not shown), but we cannot rule out the possibility of a small contamination by other single-cell clones (e.g., a heterogeneous population emerged of +/+, +/– cells).

It has previously been shown that complete genetic deletion of HuR is embryonically lethal in mouse models, and this deletion leads to lethality within 10 days (40). Interestingly, mice with conditional HuR deletion in a cell/tissue-specific manner are viable as observed in the GI tract (41, 42), lung epithelium (43, 44), brain (45), T cells (46), and macrophages (47), and suggested that deletion of HuR in GI cancer cells would be feasible. Our findings demonstrate that PDA and colorectal cancer HuR-null cells are able to establish and grow in vitro; however, when cells become stressed with chemotoxic agents (Supplementary Fig. S2A), growth in soft agar (Fig. 2D and E), and in nude mice, they were unable to survive (Fig. 4A and B; Supplementary Fig. S3A and S3B; Fig. 4C and D). In principle, the in vivo microenvironment simulates a more stressful, hypoxic, and glucose-deprived environment as compared with an in vitro environment containing high nutrient and normoxic conditions.

This work strongly supports the ongoing notion that HuR is a viable target in PDA and colorectal cancer. Currently, the best-characterized small-molecule inhibitor of HuR is the chrysanthone-like compound MS-444 that inhibits HuR homodimerization and its cytoplasmic translocation (30, 48). There have been promising preclinical studies that show MS-444 has potent effects in colorectal cancer mouse models (30), and other studies have demonstrated that MS-444 sensitizes PDA cells to oxaliplatin and 5-fluorouracil in relevant tumor microenvironment conditions, but issues with stability and bioavailability of MS-444 may hinder first human applications (17). Recently, there has been success in identifying other small molecules that disrupt the HuR–mRNA interactions, leading to impaired HuR function and premature decay of mRNAs that encode proteins related to tumor processes (37). There has also been innovative work conjugating siHuR to a 3D nanocarrier in order to target HuR in ovarian tumor–bearing mice, resulting in the suppression of tumor growth and ascites development, and ultimately prolonging lifespan (37, 39). A limitation of the models analyzed herein is the ability to assess the specificity of targeting HuR in GI cancer cells. Although previous work has shown that HuR is abundant in GI cancer cells compared with normal adjacent cells, future strategies will focus on targeting HuR expression and function specifically in cancer cells. Still, these findings indicate that posttranscriptional regulation is a promising target in cancer cells, and future studies utilizing these cell culture models of PDA and colorectal cancer should be a valuable resource for advancing the field. It is our hope that HuR inhibitors can eventually become a viable therapeutic option for patients either as a monotherapy or in combination of other current strategies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: S. Lal, E.C. Cheung, M. Zarei, C.J. Yeo, P.M. Campbell, J.M. Winter, D.A. Dixon, J.R. Brody

Development of methodology: S. Lal, E.C. Cheung, M. Zarei, R. Preet, N.C. Mambelli-Lisboa, P.M. Campbell, J.M. Winter, D.A. Dixon, J.R. Brody

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Lal, E.C. Cheung, M. Zarei, S.N. Chand, C. Romeo, M.C. Stout, A. Goetz, P.M. Campbell, D.A. Dixon

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Lal, E.C. Cheung, M. Zarei, R. Preet, S.N. Chand, N.C. Mambelli-Lisboa, C. Romeo, M.C. Stout, E. Londin, A. Nevler, P.M. Campbell, D.A. Dixon, J.R. Brody

Writing, review, and/or revision of the manuscript: S. Lal, E.C. Cheung, M. Zarei, R. Preet, S.N. Chand, C. Romeo, C.J. Yeo, P.M. Campbell, J.M. Winter, D.A. Dixon, J.R. Brody

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Lal, C.J. Yeo, J.M. Winter, J.R. Brody

Study supervision: P.M. Campbell, J.M. Winter, D.A. Dixon, J.R. Brody

Grant Support

This work was supported by the NIH [R21 CA182692 (J.R. Brody), R01 CA134609 (D.A. Dixon)], 1R01CA212600-01 (J.R. Brody), American Cancer Society MRSG-14-019-01-CDD (J.M. Winter, J.R. Brody), NIH/NCI Cancer Center Support GrantP30 CA168524 (D.A. Dixon), a Research Starter Grant Award in Translational Medicine and Therapeutics (P.M. Campbell), a grant from the W.W Smith Charitable Trust (P.M. Campbell and J.R. Brody), and fellowship awards from Prevent Cancer Foundation and K-INBRE P20 GM103418 (R. Preet). We also wish to acknowledge the Mary M. Halinski Pancreatic Cancer Research Fund; Fund A Cure; Gail V. Coleman-Kenneth M. Bruntel Charitable Grant Fund, and Research supported by the 2015 Pancreatic Cancer Action Network-AACR Research Acceleration Network Grant, grant number 15-90-25-BROD. We are also supported by the Cancer Center Support Grant5P30CA056036-17 and used the shared resource (the Core Genomics Laboratory). This work was initiated by a seed grant from the Hirshberg Foundation (J.R. Brody).

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.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

  • Received October 24, 2016.
  • Revision received November 30, 2016.
  • Accepted January 25, 2017.

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CRISPR Knockout of the HuR Gene Causes a Xenograft Lethal Phenotype
Shruti Lal, Edwin C. Cheung, Mahsa Zarei, Ranjan Preet, Saswati N. Chand, Nicole C. Mambelli-Lisboa, Carmella Romeo, Matthew C. Stout, Eric Londin, Austin Goetz, Cinthya Y. Lowder, Avinoam Nevler, Charles J. Yeo, Paul M. Campbell, Jordan M. Winter, Dan A. Dixon and Jonathan R. Brody
Mol Cancer Res June 1 2017 (15) (6) 696-707; DOI: 10.1158/1541-7786.MCR-16-0361
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