Interaction between Polyamines and the Mitogen-Activated Protein Kinase Pathway in the Regulation of Cell Cycle Variables in Breast Cancer Cells

Abstract

Inhibition of polyamine biosynthesis with A-difluoromethylornithine (DFMO) has been shown to inhibit proliferation of breast cancer cells although its mechanism of action has not been fully elucidated. To address this issue, we tested the effects of DFMO on cell cycle variables of MDA-MB-435 human breast cancer cells in culture. We also focused on the possible mediatory role of the mitogen-activated protein kinase (MAPK) pathway on the cell cycle effects of DFMO because this compound has been shown to activate MAPK signaling. We found that DFMO caused a p53-independent increase in p21 and its association with cyclin-dependent kinase (cdk)-2 and decreased cdk-2 protein as well as its phosphorylation on Thr160. In addition, DFMO markedly suppressed the expression of the full-length and low molecular weight forms of cyclin E. These effects of DFMO were reversible with exogenous putrescine, thus indicating that they are specifically mediated through polyamine depletion. Cdk-2 activity was drastically reduced in DFMO-treated breast cancer cells which exhibited a reduction in retinoblastoma (Rb) phosphorylation and protein. As a predictable consequence of these effects, DFMO caused a G 1 -S block. In addition, DFMO inhibited G 2 -M transition, most likely as a result of its induction of p21 expression. Inhibition of the MAPK pathway with PD98059 or U0126 blocked the DFMO-induced induction of p21 and the reduction of cdk-2 protein. PD98059 reversed the G 2 -M block induced by DFMO (probably as a result of suppression of p21) but not the G 1 -S arrest. MDA-MB-435 cells treated with PD98059 or U0126 in the presence and absence of DFMO exhibited a marked increase in the expression of p27 and its association with cdk-2, a decrease in phosphorylation of cdk-2 on Thr160, and a decrease in cyclin E expression. As predicted, PD98059 treatment reduced cdk-2 activity and Rb phosphorylation while reversing the decrease in Rb protein induced by DFMO. Neither DFMO nor PD98059, either alone or in combination, reduced cdk-4 activity despite a marked induction in p15 expression caused by DFMO. Our results indicate that activation of the MAPK pathway accounts for some of the effects of DFMO on cell cycle events of breast cancer cells. Inhibition of the MAPK pathway, however, does not reverse the cell cycle arrest induced by DFMO because of activation of alternative mechanisms leading to suppression of cdk-2 activity. (Cancer Res 2005; 65(23): 11026-33)

Role of non-receptor and receptor tyrosine kinases (TKs) in the antitumor action of α-difluoromethylornithine (DFMO) in breast cancer cells

“…We have observed that targeting the polyamine pathway with DFMO, an irreversible inhibitor of ODC, reduces metastasis to lungs and bones from MDA-MB-435 breast cancer xenografts in nude mice [9,10,31]. We have also observed that activation of the MAPK pathway by DFMO is causally linked to its antiinvasive effect [26], as well as to its ability to stimulate the anti-angiogenic protein thrombospondin-1 [26], to reduce the proteases meprin alpha and MMP-7 [32], and to induce G1/S cell cycle arrest [11]. In the aggregate, these results suggest that activation of the MAPK pathway may play a mediatory role in the antimetastatic effect of DFMO.…”

“…a-difluoromethylornithine (DFMO) is an irreversible inhibition of L-ornithine decarboxylase (ODC), the first and rate limiting enzyme in the polyamine synthetic pathway forming putrescine, which induces apoptosis, anti-angiogenesis, and antimetastasic effects in different cancer models [7,8]. We have consistently observed that DFMO significantly reduced proliferation, invasiveness and metastatic capacity of MDA-MB-435 breast cancer cells [9][10][11]. We have also reported that these antitumor effects are associated with activation of multiple signaling pathways, including Stat3, Stat1, JNK, and MAPK [12].…”

Evaluation of Difluoromethylornithine for the Chemoprevention of Barrett's Esophagus and Mucosal Dysplasia

“…Cyclin E expression was detected in 10% to 18% of esophageal tissues with dysplasia or adenocarcinoma, as well as in regenerative metaplastic epithelium, but was not useful in the identification of BE patients at increased risk for the development of carcinoma (29). Of note, DFMO was shown to downregulate cyclin E expression in human breast cancer cells (49). Other genes that influence cell proliferation and were modulated by DFMO include HLRC1 (MGC4293), also known as the DOHH gene (33,34), and LOC134492 whose NudCL2 product localized to the centrosome, spindle poles, and kinetochores during mitosis (35).…”

Effects of α-difluoromethylornithine on thrombospondin-1 production by human breast cancer cells

“…Furthermore, following intracardiac injection of GFP-tagged MDA-MB-435 cells, DFMO treatment significantly decreased the incidence of bone metastasis by 37% and the area of bone occupied by tumor by 65% (3). Our studies have revealed a complex interaction between ODC and multiple signal transduction pathways as well as cell cycle events which are likely related to the invasive and metastatic properties of breast cancer (3,4). Among the multiple effects of DFMO we have shown that induction of MAPK activation is instrumental in its antiinvasive action since the anti-invasive effects of the drug could be reversed with the MEK inhibitor PD98059 (5).…”