Tolfenamic acid (TA), betulinic acid (BA) and acetylsalicylic acid (aspirin) are anticancer drugs, and Sp1, Sp3, Sp4 transcription factors and growth factor 2 (EGFR2, HER2 / ErbB2) are important molecular markers in cancer cells. In this study the molecular mechanisms by which these anticancer drugs target downregulation of Sp1, Sp3, Sp4 and ErbB2 are investigated in breast cancer cells. TA inhibits growth of ErbB2-overexpressing BT474 and SKBR3 breast cancer cells by inhibiting ErbB2 expression. In cells treated with TA, ErbB2 mRNA expression and promoter activity were decreased, and this was due to decreased mRNA stability in BT474 cells. In both cell lines, TA also decreased expression of the YY1 and AP-2 transcription factors that are required for basal ErbB2 expression. These effects were accompanied by decreased ErbB2-dependent kinase activities, induction of p27, and decreased expression of cyclin D1. In addition, TA also inhibited tumor growth in BT474 cells orthotopic mouse model. However, Sp proteins were not the major target of TA in these breast cancer cells and this contrasts to results in pancreatic cancer cells where TA decreased expression of Sp proteins. BA inhibits growth of ErbB2 –overexpressing BT474 and MDA-MB-453 and induced apoptosis in these cells. BA induced proteasome-independent downregulation of specificity protein (Sp) transcription factors Sp1, Sp3, Sp4 and survivin, an Sp-regulated gene, and BA also decreased expression of ErbB2, ErbB2-regulated kinases and also YY1, a transcription factor that regulates ErbB2 expression in these cells. Knockdown of Sp1, Sp3, Sp4 and their combination by RNA interference was accompanied by decreased expression of ErbB2, YY1 and luciferase activity in cells transfected with a construct containing the GC-rich YY1 promoter linked to a luciferase reporter gene. BA-dependent repression of Sp1, Sp3, Sp4 and Sp regulated genes was due in part to induction of the Sp repressor ZBTB10 and downregulation of microRNA-27a (miR-27a) which constitutively inhibits ZBTB10 expression. The effects of BA on the miR-27a:zBTB10-Sp transcription factor axis were inhibited in cells cotreated with the cannabinoid 1 (CB1) and CB2 receptor antagonists AM251 and AM630 respectively. However, in vitro binding studies with ≤ 10 mM BA and a radiolabeled cannabinoid did not indicate direct competitive binding of BA to the CB1 and CB2 receptors, suggesting a possible role for other CB-like G protein-coupled receptors. Aspirin inhibits growth, induces apoptosis and decreases cell migration in BT474 and MDA-MB-453 breast cancer cells that overexpress the ErbB2 oncogene. Aspirin also downregulated ErbB2 expression in these cells and this was accompanied by inhibition of downstream kinases including phospho-Akt (p-Akt) and phospho-mitogen-activated protein kinase (p-MAPK). Aspirin also decreased expression of survivin, vascular endothelial growth factor (VEGF) and YY1 which regulates ErbB2 expression in these cell lines. Aspirin also downregulates Sp1, Sp3 and Sp4, and further investigation of the underlying mechanism of action showed that aspirin-induced downregulation of Sp transcription factors and Sp-regulated genes could be inhibited in part by proteasome inhibitior lactacystin and phosphatase inhibitors including sodium orthovanadate (SOV). These results were consistant with the induction of several phosphatases by aspirin in BT474 and MDA-MB-453 cells and these include mitogen-activated protein kinase phosphatase-5 (MKP-5) and MKP-1. Aspirin-induced downregulation of Sp1, Sp3 and Sp4 are reversed in cells transfected with an oligonucleotide (siMKP5) that knocks down MKP5 by RNA interference whereas siMKP-1 did not block Sp protein downregulation demonstrating for the first time a linkage between a drug-induced phosphatase (MKP-5) and Sp downregulation. These results suggest that TA, BA and aspirin represent novel and promising new anticancer drugs for cancer treatment by targeting Sp proteins and ErbB2 oncogene.