The Chromatin-Modifying Protein HMGA2 Promotes Atypical Teratoid/Rhabdoid Cell Tumorigenicity

Abstract

Atypical teratoid/rhabdoid tumor (AT/RT) is an aggressive pediatric central nervous system tumor. The poor prognosis of AT/RT warrants identification of novel therapeutic targets and strategies. High-mobility Group AT-hook 2 (HMGA2) is a developmentally important chromatin-modifying protein that positively regulates tumor growth, self-renewal, and invasion in other cancer types. High-mobility group A2 was recently identified as being upregulated in AT/RT tissue, but the role of HMGA2 in brain tumors remains unknown. We used lentiviral short-hairpin RNA to suppress HMGA2 in AT/RT cell lines and found that loss of HMGA2 led to decreased cell growth, proliferation, and colony formation and increased apoptosis. We also found that suppression of HMGA2 negatively affected in vivo orthotopic xenograft tumor growth, more than doubling median survival of mice from 58 days to 153 days. Our results indicate a role for HMGA2 in AT/RT in vitro and in vivo and demonstrate that HMGA2 is a potential therapeutic target in these lethal pediatric tumors.

Introduction

Atypical teratoid/rhabdoid tumors (AT/RTs) are highly aggressive and deadly pediatric brain tumors with a very poor prognosis ( 1 ). Current therapies of surgery, radiation, and intense chemotherapy allow only a very small subset of children to survive ( 2 ). Exposing infants and young children to very aggressive therapy also leads to lifelong cognitive, behavioral, and growth deficits in survivors ( 3 ).

The sole consistently identified genomic alteration in AT/RTs is in the chromatin remodeling complex gene SMARCB1/INI1/SNF5 ( 4, 5 ). Loss of the tumor suppressor INI1 blocks proper differentiation of neural stem and progenitor cells and is believed to be critical for the development of AT/RTs ( 6 ). Therapeutic failure in aggressive brain tumors such as AT/RTs is caused by the lack of potency of existing agents, the impermeability of the blood–brain barrier, intratumoral and intertumoral heterogeneity, and activation of antiapoptotic and metabolic programs that allow tumor cells to survive treatment ( 7, 8 ). Identification and validation of novel targets are essential to develop better therapies and improve the dismal prognosis of this lethal pediatric tumor.

Atypical teratoid/rhabdoid tumors share many characteristics with stem cells, including an ability to differentiate into cells with neuronal and “rhabdoid” features, as well as resistance to chemotherapy and radiation ( 1, 9 ). Atypical teratoid/rhabdoid tumors express multiple cell factors, including SOX2, NANOG, KLF4, and High-mobility Group AT-hook 2 (HMGA2) ( 10, 11 ). HMGA2 is a chromatin-architectural protein that is highly expressed during embryogenesis, with little to no expression in normal adult tissues ( 12–16 ). Increased expression of HMGA2 is associated with a poor prognosis in multiple adult cancer types, including lung, gastric, pancreatic, and ovarian carcinomas and leukemia ( 11, 17–26 ). High-mobility Group AT-hook 2 promotes tumor cell growth, invasion, and clonogenic potential in cancer cells ( 13, 14, 17–21, 27–33 ). Reduction of HMGA2 in a kidney RT cell line decreased proliferation and colony formation ( 11 ), but the functional significance of HMGA2 in central nervous system (CNS) AT/RTs and the role of HMGA2 in CNS AT/RT tumor formation in vivo are unknown.

We here show that HMGA2 is expressed in CNS AT/RT cell lines derived from pediatric patients. Short-hairpin RNA (shRNA)–mediated knockdown of HMGA2 in these AT/RT cell lines suppressed growth, proliferation, and colony formation in vitro. Knockdown of HMGA2 increased apoptosis in vitro and increased tumor latency in vivo. Our studies demonstrate the functional importance of HMGA2 in regulating multiple transformed properties of AT/RTs and suggest that targeting HMGA2 may be a valid therapeutic approach in this aggressive tumor.

Materials and Methods

Cell Lines and Cell Culture

The BT37 AT/RT cell line was derived from a human xenograft that originated at St. Jude Children’s Research Hospital (Memphis, TN) and was passaged serially in immunodeficient mice. The tumor tissue was minced and suspended in RPMI-1640 medium containing penicillin (100 U/mL), streptomycin (100 μg/mL), and 20% fetal bovine serum (FBS). The cultures were incubated at 37°C in a humidified atmosphere of 5% CO ₂ . The medium was changed every 4 to 5 days. On reaching the confluent state, the monolayers were treated with trypsin and the dispersed cells were transferred into new culture flasks. Cells were acclimated to growth as semiadherent cells in 10% FBS/RPMI-1640, 1% penicillin/streptomycin. CHLA-02-ATRT, CHLA-04-ATRT, CHLA-05-ATRT, and CHLA-06-ATRT cell lines were generated from pediatric AT/RT tumor samples obtained from Children’s Hospital of Los Angeles (Los Angeles, CA). Tumor tissue was prepared within 30 to 60 minutes as described ( 34 ). Cells were initially cultured as neurospheres in modified Neurobasal medium consisting of 1:1 DMEM:F12 containing 15 mmol/L HEPES, 110 mg/L sodium pyruvate, 1.2 g/L sodium bicarbonate, B27 supplement (Gibco, Grand Island, NY), 20 ng/mL epidermal growth factor (Peprotech, Inc, Rocky Hill, NJ), 20 ng/mL basic fibroblast growth factor (Peprotech), and 25 μg/mL gentamicin (Gibco). Gentamicin was removed after the first 2 weeks of culture. Passaging was at a ratio of 1:2–3, with 25% (vol/vol) conditioned medium in the new flask. CHLA-05-AT/RT and CHLA-06-AT/RT were originally described in Erdreich-Epstein et al ( 35 ). Details of the cell lines are described in the Table, Supplemental Digital Content 1 ( Supplementary Data ). All the AT/RT cell lines were authenticated using short tandem profiling using StemElite kit (Promega, Madison, WI) at the Genetic Resources Core Facility in The Johns Hopkins University. Eight short tandem repeat loci along with a gender-determining marker, Amelogenin, were used to authenticate the BT37 cell line (Table, Supplemental Digital Content 2, Supplementary Data ). CHLA-02 and CHLA-04 are available from the American Type Culture Collection ([ATCC] Manassas, VA). BT-12 is available from the Children’s Oncology Group cell line and xenograft repository ( 36, 37 ). All of the AT/RT cell lines used in this article were authenticated to be human and did not match the short tandem repeat profile of any other cell line in the established databases (ATCC, DSMZ, and JCRB). 293 T cells were used for the production of lentiviral particles and were cultured in DMEM containing 10% FBS and were obtained through ATCC. The SF188 cell line was used as a positive control for the expression of INI1 and was cultured in DMEM/F12 supplemented with 10% FBS ( 38 ).

Lentiviral Knockdown of HMGA2

Two lentiviral MISSION TRC (Human) shRNA constructs were used to reduce HMGA2 mRNA expression: TRCN0000342671 (targets the coding region, denoted as shHMGA2-2) and TRCN0000342673 (targets the 3′-UTR, denoted as shHMGA2-1). MISSION pLKO.1-puro Empty Vector Control was used as control shRNA (denoted as shCTL) in all experiments. For producing lentiviruses, 293 T cells were transfected with 0.3, 2.5, and 3 μg of VSVG envelope plasmid, delta 8.9 gag/pol plasmid, and the lentiviral plasmid of interest, respectively, using Fugene 6 (Promega). Viral supernatants were pooled after 48, 72, 96, and 120 hours posttransfection and filtered through a 0.45-μm filter. Virus was concentrated using 5% PEG-8000 and 0.15 mol/L NaCl and resuspended in 500 μL of DMEM. Small aliquots of concentrated virus were stored at −80°C until further use. For knocking down HMGA2, concentrated virus was added to confluent cultures of BT37 and CHLA-06 cells. After 48 hours, transduced cells were selected using puromycin (1–2 μg/mL; Sigma-Aldrich, St. Louis, MO) for 5 days. Reduced expression of HMGA2 in puromycin-selected cells was confirmed using immunoblotting. All experiments were performed within 14 days of infecting AT/RT cells with shRNA lentivirus.

Immunoblotting

Cells were harvested and washed in ice-cold 1× PBS (Life Technologies, Grand Island, NY). Cell pellets were lysed, separated on polyacrylamide gels, and immunoblotted as described ( 39 ). INI1, HMGA2, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, and cleaved poly (ADP-ribose) polymerase (PARP) expression were analyzed using the antibodies to the following: INI1 (Sigma-Aldrich), HMGA2 (Cell Signaling Technology, Inc, Beverly, MA), GAPDH (Research Diagnostics, Inc, Flanders, NJ), β-actin (Santa Cruz Biotechnology, Inc, Dallas, TX), and cleaved PARP (Cell Signaling Technology, Inc). Primary antibodies were detected using goat anti-mouse and goat anti-rabbit antibodies conjugated to horseradish peroxidase (KPL, Gaithersburg, MD). Secondary antibodies were detected using Western Lightning Plus ECL (PerkinElmer, Waltham, MA). Densitometry analysis was performed using ImageJ software ( 40 ).

Cell Growth and Proliferation Assays

For assessing differences in cell growth, 2,500 transduced single cells were plated in triplicate in 96-well plates. Cell growth was measured on Day 1 (day of plating cells), Day 3, and Day 5 using the colorimetric CellTiter 96AQueous One Solution Assay (MTS, Promega). For measuring cell proliferation, transduced cells were pulsed with bromodeoxyuridine (BrdU; Sigma-Aldrich) for 6 hours and then processed for immunofluorescence as described ( 39 ).

Apoptosis Assays

For measuring apoptosis, transduced and selected cells were cytospun onto positively charged slides and processed for immunofluorescence for detecting cleaved caspase-3. Immunoblotting was performed to detect cleaved PARP as previously described.

Immunofluorescence

Immunofluorescence was performed as previously described ( 39 ) with primary antibodies to the following: BrdU (Sigma-Aldrich) and cleaved caspase-3 (Cell Signaling Technology).

Colony Formation Assay

Infected and selected cells were plated in soft agarose to form colonies as previously described ( 39 ) with the following modifications: media were changed every 2 days and the cultures were incubated for 2 weeks.

Intracranial Tumor Xenografts

Freshly infected BT37 cells expressing either control or HMGA2 shRNA were selected under puromycin for 5 days. After knockdown was verified by immunoblotting, 100,000 viable cells were injected into the right striatum of nude mice as previously described ( 39 ). Mice were monitored daily and were killed when they showed signs of tumor formation (neurologic impairment, weight loss, hunching) in accordance with Institutional Animal Care and Use Committee–accepted protocols.

Immunohistology

Brains were removed and placed into 10% formalin for 48 hours, after which they were embedded in paraffin and sectioned by the Johns Hopkins histology core facility.

Survival Analysis and Statistical Methods

Kaplan-Meier survival graphs and logrank tests were generated for the in vivo experiments using GraphPad Prism (GraphPad Software, Inc, La Jolla, CA). Statistical tests for all other experiments were performed using Excel (Microsoft). Results are shown for each experiment performed independently at least twice with concordant results. For in vivo experiments, data were analyzed from at least 4 animals injected per condition. Error bars denote SE of experiments performed independently at least twice. Significance of the experimental results was determined by an unpaired 2-sided Student t -test, where p < 0.05 was considered to be significant.

Results

The BT37 Cell Line Forms Aggressive Orthotopic Xenograft Tumors Pathologically and Clinically Similar to AT/RT

We transitioned the BT37 AT/RT serially passaged human xenograft tumor to a rapidly growing semiadherent cell line. Injection of BT37 into the brains of immunodeficient mice led to aggressive xenograft tumor formation, with most mice succumbing within 60 days of injection. Examination of orthotopic xenografts revealed the presence of multiple mitotic figures, apoptosis, and the presence of classic RT cells ( Fig. 1 A, B).

Atypical teratoid/rhabdoid tumors are characterized by chromosomal mutations or deletions in the hSNF5/INI1 gene, leading to loss of INI1 protein expression ( 41, 42 ). BT37, along with other AT/RT cell lines, did not express the INI1 protein, as shown by immunoblotting ( Fig. 1 C).

AT/RT Cell Lines Express HMGA2

When we tested the expression of HMGA2 protein in our AT/RT cell lines by immunoblotting, we found that 5 of 6 AT/RT cell lines express HMGA2 ( Fig. 1 D) protein. To investigate the role of HMGA2 in AT/RT directly, we used shRNA to suppress HMGA2 expression. CHLA-06 and BT37 cell lines were infected with lentivirus encoding either control or HMGA2 shRNA. Infected cells were selected with puromycin for 5 days and tested for HMGA2 expression by immunoblotting ( Fig. 1 E). Densitometry analysis of immunoblots depicted an average reduction of 59% and 52% for shHMGA2-1 and -2 in CHLA06 and 77% and 59% for shHMGA2-1 and -2 in BT37 compared with control shRNA.

Knockdown of HMGA2 Reduces Growth and Proliferation of AT/RT Cells

Atypical teratoid/rhabdoid tumor is a rapidly growing tumor of the CNS ( 1 ). Because expression of HMGA2 is associated with increased growth and proliferation ( 17, 18, 29, 32 ), we hypothesized that HMGA2 promotes the growth and proliferation of AT/RT cells. We found that shRNA knockdown of HMGA2 reduced growth of CHLA-06 and BT37 cells compared with control shRNA-expressing cells ( Fig. 2 A; p < 0.05 for shHMGA2-1 for both cell lines, with concordant but not statistically significant results using shHMGA2-2). To investigate the mechanism of growth inhibition, we performed BrdU incorporation assay to determine if HMGA2 suppression would inhibit AT/RT proliferation. Reduction of HMGA2 significantly inhibited the proliferation of CHLA-06 and BT37 cells compared with control shRNA transduced cells, as measured by BrdU uptake ( Fig. 2 B, C; p < 0.05 for shHMGA2-1 and -2).

Suppression of HMGA2 Increases Apoptosis of AT/RT Cells

Another potential mechanism by which HMGA2 loss could affect AT/RT growth is by inducing apoptosis. HMGA2 is known to prevent apoptosis in multiple tumor types ( 43–45 ). During apoptosis, caspase-3 enzyme is activated because of proteolytic cleavage ( 46 ). Cleaved caspase-3 (CC3) then cleaves and activates PARP ( 47 ). Cleaved caspase-3 and cleaved PARP are well-accepted markers to denote cells undergoing apoptosis. We determined the induction of CC3 by immunofluorescence in CHLA-06 and BT37 cells after lentiviral transduction with either control or HMGA2 shRNA. Knockdown of HMGA2 in CHLA-06 cells increased CC3 immunopositivity by an average of 2.6- and 2.2-fold for shHMGA2-1 and -2, respectively, compared with control shRNA ( Fig. 3 A, B; p < 0.05 for shHMGA2-1 and -2).Knockdown of HMGA2 in BT37 led to a 9.1- and 9.8-fold average increase in CC3 immunopositivity for shHMGA2-1 and -2, respectively, compared with control shRNA ( Fig. 3 A, B; p < 0.05 for shHMGA-1 and -2). Similarly, there was an increase in cleaved PARP expression in HMGA2 shRNA–transduced cells compared with control cells as assessed by immunoblotting ( Fig. 3 C). These results suggest that HMGA2 prevents apoptosis to promote growth of AT/RT cells in vitro.

HMGA2 Suppression Prevents Clonogenic Growth of AT/RT Cells

We plated single cells of CHLA-06 expressing either control or HMGA2 shRNA in soft agarose ( Fig. 4 A, B). After 2 weeks, cells expressing control shRNA formed an average of 82 ± 4 colonies. In comparison, cells expressing HMGA2 shRNA formed only 40 ± 4 (for shHMGA2-1; p < 0.01) and 62 ± 5 (for shHMGA2-2; p < 0.05) colonies ( Fig. 4 A, B).

Knockdown of HMGA2 Decreases Tumor Formation In Vivo

To explore whether HMGA2 is required for tumor formation in an in vivo orthotopic environment, we injected BT37 cells expressing either control or HMGA2 shRNA into the right striatum of immunodeficient mice. Animals were monitored regularly for signs of an increasing tumor mass and sacrificed on reaching a moribund state. Logrank analysis of Kaplan-Meier survival curves showed that the cohort of mice receiving control cells (n = 4) survived for a median of 58 days, whereas mice injected with HMGA2-deficient cells (n = 8) lived longer (median survival, 153 days; p = 0.0004) ( Fig. 4 C). These results indicate that knockdown of HMGA2 significantly increased the latency period of tumor formation and led to increased survival of orthotopically xenografted mice, signifying the importance of HMGA2 for AT/RT tumor growth in brain environment. Histologic examination of tumors showed no clear differences between tumors that formed from HMGA2-deficient BT37 cells compared with control BT37 cells.

Discussion

The hallmark alteration in AT/RT is the loss of the INI1 chromatin-modifying protein, which may function to promote differentiation ( 5, 6 ). Maintenance of an undifferentiated state and persistence of factors that promote self-renewal, invasion, and proliferation likely contribute to AT/RT tumorigenicity. HMGA2 is a regulator of normal neural stem cell renewal ( 30 ). Our finding that inhibition of HMGA2 suppresses AT/RT cell growth is concordant with those showing that HMGA2 loss blocks proliferation of malignant RT cell lines ( 11 ). The suppression of AT/RT orthotopic xenograft formation after HMGA2 inhibition suggests that targeting HMGA2 directly may be a useful therapeutic modality in AT/RT and malignant RT.

High-mobility group A2 is a chromatin-modifying protein that binds to the minor groove of DNA at multiple sites in the genome ( 12, 14, 48–51 ). The exact mechanism by which HMGA2 promotes self-renewal, proliferation, and invasion in multiple tumor types remains largely unknown. The wide range of promoters and enhancers that HMGA2 can bind to makes it an attractive driver of AT/RT tumorigenesis because, similar to INI1, HMGA2 can influence thousands of genes in the genome. In addition to binding directly to enhancers and activating or suppressing transcription, HMGA2 is also known to bind to and stabilize RNA molecules ( 24 ). Continued expression of HMGA2 in addition to INI1 may allow AT/RT tumor cells to regulate thousands of tumorigenic genes and provides an attractive mechanism to explain the aggressive nature of AT/RT in the absence of multiple recurrent activating genetic mutations.

Netropsin and other drugs that bind in the minor groove of DNA, including Hoechst 33258, inhibit the binding of AT-hook–containing proteins to their consensus sites ( 52–55 ). Netropsin can suppress the growth of HMGA1-expressing medulloblastoma cells ( 56 ). The data presented here show that targeting HMGA2 may be therapeutically beneficial in AT/RT.

Acknowledgment

The authors thank Sabeen Zulfiqar Ali for technical assistance with immunofluorescence experiments and Dr Peter Houghton for the kind gift of the BT37 AT/RT xenograft.

References

Supporting Information

Supplementary Data

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Author notes