Inhibition of rhabdomyosarcoma cell and tumor growth by targeting specificity protein (Sp) transcription factors

Abstract

Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are highly expressed in rhabdomyosarcoma (RMS) cells. In tissue arrays of RMS tumor cores from 71 patients, 80% of RMS patients expressed high levels of Sp1 protein, whereas low expression of Sp1 was detected in normal muscle tissue. The non-steroidal anti-inflammatory drug (NSAID) tolfenamic acid (TA) inhibited growth and migration of RD and RH30 RMS cell lines and also inhibited tumor growth in vivo using a mouse xenograft (RH30 cells) model. The effects of TA were accompanied by downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes in RMS cells and tumors, and the role of Sp protein downregulation in mediating inhibition of RD and RH30 cell growth and migration was confirmed by individual and combined knockdown of Sp1, Sp3 and Sp4 proteins by RNA interference. TA treatment and Sp knockdown in RD and RH30 cells also showed that four genes that are emerging as individual drug targets for treating RMS, namely c-MET, insulin-like growth factor receptor (IGFR), PDGFRα and CXCR4, are also Sp-regulated genes. These results suggest that NSAIDs such as TA may have potential clinical efficacy in drug combinations for treating RMS patients.

Figure 1

Sp protein expression in RMS cells and tumors and effects of TA. (A) RH30 and RD cells. Cells were treated with DMSO or TA, and whole cell lysates were examined for expression of Sp1, Sp3 and Sp4 proteins by western blots as described in the Materials and Methods. (B) Genetic models of RMS. Whole cell lysates from HSMM and stably transfected RMS cells were analyzed for protein expression as described in (A). Relative intensity of Sp1 staining in RMS patient tumors and muscle tissue (C) and in specific samples (D). Samples provided by the Cooperative Human Tissue Network were stained for Sp1 and relative staining intensities were assigned as described in the Materials and Methods. β-Actin was used as a loading control for all western blots.

Figure 2

TA inhibits RMS cell growth, induces apoptosis, and inhibits migration. Effects of TA on cell proliferation (A) and cell cycle progression (B). RH30 and RD cells were treated with DMSO, TA or TA+IGF (100 ng/ml) (cell proliferation only) , and effects on cell proliferation or percent distribution of cells in G₀/G₁, S and G₂/M phases were determined as described in the Materials and Methods. Induction of apoptosis (C) and inhibition of RMS cell migration by TA (D). RMS cells were treated with DMSO, TA or TA+IGF (100 ng/ml), and effects on Annexin V staining and cell migration (scratch assay) were determined as outlined in the Materials and Methods. Results are expressed as means ± SE for at least 3 separate determinations, and significant (p < 0.05) induction (*) or inhibition (**) by TA is indicated.

Figure 3

Effects of RNAi and TA on Sp1, Sp3, Sp4 and Sp-regulated genes in RMS cells. (A) Downregulation of Sp proteins by RNAi. Cells were transfected with specific oligonucleotides or a combination, and whole cell lysates were analyzed by western blots as described in the Materials and Methods. TA decreases CD1, survivin, VEGF (B) and other critical gene products (C) in RMS cells. Cells were treated with DMSO or TA, and whole cell lysates were analyzed by western blots as described in the Materials and Methods. (D) Effects of RNAi on Sp-regulated genes. RMS cells were transfected with oligonucleotides as outlined in (A), and whole cell lysates were analyzed by western blots as described in the Materials and Methods.

Figure 4

TA and RNAi inhibit RMS cell migration (Boyden chamber) and proliferation. Inhibition of RH30 (A) and RD (B) cell migration by TA. RMS cells were treated with DMSO or TA, and inhibition of cell migration in the Boyden chamber assay was determined as outlined in the Materials and Methods. Knockdown of Sp and effects on RMS cell migration (C) and proliferation (D). Cells were transfected with iSp1 and effects on cell migration (Boyden chamber) and effects of iSp1, iSp3, iSp4 and iSp1/3/4 on proliferation were determined as described in the Materials and Methods. Results in (A)-(D) are means ± SE for at least 3 separate experiments, and significant (p < 0.05) inhibition by TA or Sp knockdown are indicated (*). The DMSO and iLamin treatment controls were set at 100%, and the absolute values for these controls in RH30 and RD cells were not significantly different.

Figure 5

Activation of proteasome-dependent or -independent pathways by TA. Effects of proteasome inhibitors on TA-induced Sp downregulation in RH30 (A) and RD (B) cells. Cells were treated with DMSO or TA in the presence or absence of proteasome inhibitors, and expression of Sp proteins in whole cell lysates was analyzed by western blots. (C) Modulation of ZBTB10 and miR-27a expression by TA. Cells were treated with DMSO or TA for 24 hr and analyzed for expression of ZBTB10 and miR-27a by real time PCR as outlined in the Materials and Methods. Results are expressed as means ± SE for 3 separate experiments, and significant (p < 0.05) induction (*) or inhibition (**) compared to DMSO control are indicated. (D) ZBTB10 overexpression. Cells were transfected with ZBTB10 expression plasmid, and whole cell lysates were analyzed by western blots as described in the Materials and Methods.

Figure 6

TA inhibits tumor growth and downregulates Sp and Sp-regulated proteins in tumors. TA-mediated inhibition of tumor volume and weight (A) and downregulation of Sp proteins (B). Athymic nude mice bearing RH30 cells as xenografts were treated with corn oil (control) or TA (50 mg/kg/dose) (8 mice per treatment group) every second day, and tumor areas and weights were determined. Similar results were observed in a pilot study using RD cells as xenografts (3 animals per treatment group) (A). Expression of Sp-proteins and Sp-regulated genes were determined in tumor lysates from control and treated mice by western blots as outlined in the Materials and Methods. Immunostaining for Sp1 and c-MET (C) and VEGF (D). Fixed tumor samples from control and treated mice were immunostained for Sp1, c-MET and VEGF as described in the Materials and Methods. Significant (p < 0.05) inhibition of tumor growth and weights are indicated (*).