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.

Figure 1

The BT37 cell line formed aggressive orthotopic xenograft tumors with an atypical teratoid/rhabdoid tumor (AT/RT) phenotype. (A) 200× magnification showing BT37 xenograft (right) in a normal brain (left). (B) 200× view showing high-grade features such as multiple mitotic figures (black arrow), apoptotic bodies (red arrow) and “rhabdoid” cells, which are classic for AT/RT (black arrowhead). (C) Immunoblot for INI1 shows retained INI1 expression in the pediatric glioblastoma cell line SF188 and loss of INI1 in AT/RT cell lines. Immunoblots were stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Molecular weights are shown at left in kDa. (D) Immunoblot of AT/RT cell lines BT12, BT37, CHLA-02, CHLA-04, CHLA-05 and CHLA-06 showing expression of high mobility group A2 protein (HMGA2). Immunoblots were stripped and reprobed for GAPDH as a loading control. (E) Immunoblot showing knockdown of HMGA2 by 2 different lentiviral short hairpin RNAs in BT37 and CHLA06 AT/RT cell lines. Control lentiviral short hairpin RNA (shCTL) or HMGA2 shRNA (shHMGA2#1 and shHMGA2#2) were analyzed for HMGA2 expression by immunoblotting; β-actin was used as a loading control. HMGA2 and β-actin protein levels were quantitated on the immunoblot using densitometry and values normalized to control (shCTL) for each cell line.

Figure 2

Knockdown of high mobility group A2 protein (HMGA2) reduced growth and proliferation of atypical teratoid/rhabdoid tumor (AT/RT) cells in vitro. We infected AT/RT cell lines with lentiviral pLKO empty vector or HMGA2 targeting lentiviral short hairpin RNA (shRNA), selected cells with puromycin for 5 days, verified knockdown of HMGA2 by western blot, and performed assays of growth and proliferation. Control shRNA and HMGA2 shRNA are depicted as shCTL and shHMGA2 (#1 or #2), respectively. (A) HMGA2 knockdown inhibited growth of AT/RT cells. CHLA-06 and BT37 cells expressing HMGA2 shRNA showed reduced growth at day 3 and 5 compared to control shRNA by MTS assay (p < 0.05 for shHMGA2#1 compared to shCTL, n = 2 for CHLA-06, n = 3 for BT37). (B) HMGA2 knockdown reduced proliferation of AT/RT cells. Representative photomicrographs (400x magnification) of HMGA2 shRNA expressing CHLA-06 and BT37 cells depicting reduced bromodeoxyuridine (BrdU) incorporation compared to control shRNA by immunofluorescence. DAPI was used to stain all nuclei. (C) Quantitation of BrdU incorporation. The percentage of BrdU positive nuclei in shHMGA2 compared to shCTL is depicted (* and **, p < 0.05 for shHMGA#1 and #2, compared to shCTL, for both CHLA-06 [n = 2] and BT37 [n = 3]).

Figure 3

Knockdown of high mobility group A2 protein (HMGA2) increased apoptosis of atypical teratoid/rhabdoid tumor (AT/RT) cells. (A) HMGA2 knockdown increased cleaved caspase-3. Representative photomicrographs (400× magnification) showing increased cleaved caspase-3 staining in CHLA-06 and BT37 cells infected with HMGA2 lentiviral short hairpin RNA (shRNA) compared to control shRNA by immunofluorescence. (B) Quantitation of cleaved caspase-3 immunopositivity after HMGA2 knockdown. The percentage of cells positive for cleaved caspase-3 out of the total number of cells (DAPI was used to stain all nuclei) are shown (* and **, p < 0.05 for shHMGA#1 and #2, compared to shCTL, for both CHLA-06 [n = 3] and BT37 [n = 3]). (C) Knockdown of HMGA2 increased cleaved poly (ADP-ribose) polymerase (PARP) expression. CHLA-06 and BT37 cells infected with either control or HMGA2 shRNA were analyzed for cleaved PARP protein expression by immunoblotting. β-actin was used as a loading control. Cleaved PARP and β-actin protein levels were quantitated on the immunoblot using densitometry and values normalized to control shRNA (shCTL) for each cell line are depicted.

Figure 4

High mobility group A2 protein (HMGA2) knockdown reduces colony formation and tumorigenicity of atypical teratoid/rhabdoid tumor (AT/RT) cells. (A) Representative photographs of nitroblue tetrazolium-stained colonies formed by CHLA-06 cells expressing control lentiviral short hairpin RNA (shRNA) or HMGA2 shRNA. (B) Quantitation of number of colonies depicted in (A), showing reduced colony formation in CHLA-06 cells infected with HMGA2 shRNA (#1 and #2) vs. control shRNA (*p < 0.01 for shHMGA#1 [n = 3] and **p < 0.05 for shHMGA2#2 [n = 2]). (C) Knockdown of HMGA2 increased survival. Kaplan-Meier survival curves showing increased survival of mice that were injected intrastriatally with HMGA2 shRNA infected BT37 cells compared to control infected cells (p < 0.0004, Log-rank test). Data were collected from at least 4 animals per condition (n = 4 for shCTL and n = 8 for shHMGA2#1).