DEGUELIN ACTION IS THROUGH REGULATION OF ERK-MAPK PATHWAY IN TRIPLE- NEGATIVE BREAST CANCER CELLS

Breast cancer is the most common cancer and the leading cause of cancer related deaths amongst women in the United States. In 15-20% of patients with breast cancer, patients do not express steroid receptors(ER/PR) and do not have amplification of HER2. This condition is called triple negative breast cancer (TNBC). Patients with TNBC have poor prognosis. No molecular targeted therapies are available to treat TNBC. Anthracyclines and taxane based therapies are effective with limited success, and are often toxic. As TNBC cells overexpress Epidermal Growth Factor Receptor (EGFR), it provides an attractive molecular tool for the targeted therapy. Deguelin, a rotenoid isolated from an African plant, Mundulea sericea has been shown to inhibit growth of various experimental cancers. Deguelin is a well known inhibitor of pAKT, a downstream target of EGFR. In this study we evaluated the effect of deguelin and its mechanism of anticancer action in triple negative human breast cancer cell lines. Deguelin inhibited cell proliferation of MDA-MB-231, MDA-MB-468, BT-549, and BT- 20 in a dose and time-dependent manner. Western blots and immunofluorescence analyses suggested that the effect of Deguelin is mediated through inhibition of Receptor Tyrosine Kinases (EGFR, c-Met) and its downstream molecular targets, Our results show that deguelin treatment reduces the expression of pERK (p44/42) (Thr202/Tyr204), pAKT (ser473), c-Myc, pSTAT3 (Tyr705) and survivin in a dose and time dependent manner in MDA-MB-231, MDA-MB-468 human breast cancer cells. In conclusion, results from this study suggest that Deguelin may be of potential therapeutic value in treatment of triple negative breast cancers.M.S. in Biology, December 201

Deguelin action involves c-Met and EGFR signaling pathways in triple negative breast cancer cells.

Treatment of breast cancer patients with antiestrogens and aromatase inhibitor(s) or Herceptin have shown significant success in steroid receptor positive or Her-2+ breast cancers respectively. However, choice of treatments for breast cancer patients with negative status for estrogen, progesterone receptors and HER2/neu is limited. As a result, search for appropriate therapy regimen for these triple negative breast cancers (TNBC) has become a major focus of investigations for many laboratories. Recently, Deguelin, a natural product isolated from African plant Mundulea sericea (Leguminossae) has shown both antiproliferative actions in various cancers including breast as well as chemoprenventive activity against carcinogen induced experimental cancers. In this report we evaluated efficacy and mechanism of action of Deguelin in triple negative breast cancer cell lines.In vitro, Deguelin in a dose and time dependent manner inhibited the growth of MDA-MB-231, MDA-MB-468, BT-549 and BT-20 cells. Deguelin (2 or 4 mg/kg body weight), when injected intraperitoneally, reduced the in vivo tumor growth of MDA-MB-231 cells transplanted subcutaneously in athymic mice. Moreover it was nontoxic as evident from daily observations on mobility, food and water consumption and comparison of bodyweight and other visceral organ weights with those in control animals at the termination of the study. The western blot analyses and immunostaining studies indicated that the deguelin effects may be mediated through EGFR-PAKT/c-Met p-ERK and NF-κB by down regulating their downstream targets such as p-STAT3, c-Myc, Survivin.These results suggest that Deguelin may have a significant therapeutic value for the treatment of TNBC patients

Effect of Deguelin on the growth and expression of signaling molecules in TNBC cells.

<p>A. Effect of deguelin on MDA-MB 231 cells in the presence of EGF (1 ng or 20 ng) alone, Deguelin alone (31–500 nM)or Deguelin+EGF. The cells were plated in six well plates, allowed to attach overnight, then treated with indicated test compound/(s) (in serum free media) and incubated for 72 h at 37°C. Number of cells was counted in each well using coulter counter. Data represent % cells in the respective control treatment. * indicates significant difference as compared to respective control, ** indicates significant difference (p<0.05) between the groups shown. B. Effect of Deguelin on total (after permeabilization)and cell surface (without permeabilization) EGFR expression in the presence/absence of indicated concentration of EGF. The cells plated on glass coverslips were incubated for 10 min-24 h at 37°C in the serum free media containing vehicle only, Deguelin (250 nM) or Deguelin+EGF as indicated. At the end of incubation cells were fixed in 4% buffered formalin, permeabilized and then processed for immunofluorescence examination of EGFR (red) and mounted in DAPI (blue) containing media. Representative photographs captured using fluorescence microscope equipped with appropriate filters are shown. Red =  EGFR expression, Blue =  DAPI staining of nuclei. Data are shown for 2 and 24 h post treatments. C. Effect of Deguelin, EGF and EGF+Deguelin on EGFR/1045-phospho EGFR and 1068-Phospho EGFR levels in MDA-MB-231 cells. MDA-MB-231 cells were treated for 10 min–24 h with indicated treatments in serum free media as described above. At the end of incubation cells lysates were prepared and subjected to western blot analysis. For loading control appropriate control proteins were developed using appropriate antibodies. Lanes 1 = control, 2 =  Deguelin 250 nM, 3 = EGF1 ng 4 =  EGF 1 ng +Deguelin 250 nM, 5 =  EGF 20 ng 6 = EGF20 ng+Deguelin 250 nM. Number under each lane indicate % of normalized (protein intensity/control protein intensity) specific band intensity in relation to control treatment. Control was considered as 100%. D. Western blot and Immunofluorescence analyses of p-ERK and Survivin in MDA-MB-231 cells treated for 24 h with vehicle only, Deguelin (250 nM) in the presence or absence of EGF. Serum free media was used for this study. Lane 1 =  Control, 2 =  Deguelin 250 nM, 3 = 1 ng EGF, 4 = 1 ngRGF+Deguelin250 nM, 5 = 20 ng EGF, 6 = 20 ng EGF+Deguelin. For western blot number under each band indicates % of intensity of normalized values as described above. In immunofluorescence images, green fluorescence indicates p-ERK, red immunofluorescence indicates Survivin and Blue staining is due to nuclear counterstaining with DAPI. Representative images are shown. E. Western blot analysis of various cell signaling molecules (p-ERK, ERK, p-STAT-3, c-Myc) in MDA-MB-231 cells treated for 24 h with varying concentration of Deguelin in serum free media. Intensity of specific band is normalized in relation to loading control protein intensity, ratio in vehicle treated control with housekeeping protein control was considered as 100%. Number under each band indicate % of control intensity.</p

Effect of Deguelin on in vivo growth of MDA-MB-231 cells.

<p>A. MDA-MB-231 cells (3 million cells/animal) were injected s.c. in the dorsal flank of 6–7 weeks old female athymic mice. Animals were monitored for the growth of tumor at the injection sites. Once the tumor reached 50 mm³ animals received vehicle only or Deguelin (4 mg/kg bodyweight) daily by i.p. route. Tumor size was determined twice/weekly using calipers. Data are shown as mean±SE tumor volume (mm³) in each group consisting of n = 10 animals. * indicates statistical difference (p<0.05) between control and treatment group at the indicated time. B. Effect of Deguelin on body visceral organ weights: Animals treated with vehicle/Deguelin as described above, at termination body weights were recorded, lung, liver, kidney and spleens were excised at termination of experiment and weighed. Data in the bar graph represent mean+SE (weights in g) of 10 values in each group. * indicates significant difference between respective control and treatment. C. Immunohistochemical staining of PCNA, EGFR, c-Met, p-ERK, p-AKT, NF-KB (p65) and Survivin proteins in MDA-MB-231 xenografts: Paraffin embedded sections of MDa-MB-231 xenografts from vehicle and Deguelin treated animals were processed for immunohistochemical staining of indicated protein. Brown = specific protein staining, Blue =  counterstaining of nuclei with hematoxylin. Representative images taken at 40× objective are shown.</p

Figure 3

<p><b>Effect of Deguelin on EGFR, c-Met and other downstream target proteins in MDA MB 231 cells.</b> A. Effects of deguelin on the signaling molecules (EGFR, c-MET, p-ERK, ERK, p-STAT3, STAT3, c-MYC, Survivin) in the presence of 10% FBS. MDA-MB-231 cells were treated with indicated concentration of Deguelin or vehicle alone for 24–72 h in the media containing 10% serum. The cell lysates were processed for western blots. Representative western blots are shown. Note: Same Western blot membrane was processed for multiple proteins, this is reflected by same banding pattern of loading control protein. Numbers under each band show % of control of normalized (specific protein/loading control protein) pixel intensity. Values in the respective control were considered as 100%. 1 = Control; 2 = Deguelin (250 nM) treated. B. Effect of Deguelin on expression of various proteins in MDA-MB-231 cells. Cells were plated on glass coverslips, treated with Deguelin (250 nM) or vehicle only for 24, 48 h, serum containing media was used, immunofluorescence analysis was done as described above. Red/green fluorescence = specific protein expression as labeled, blue = DAPI staining for nuclei. C. Effect of vehicle or Deguelin in other breast cancer cell lines. Cells (MDA-MB-231-lanes 1,2; MDA-MB-468 lanes 3,4 and BT-549 lanes 5,6) were treated with vehicle only (lanes 1,3,5) or 250 nM Deguelin (lanes 2,4,6) for 48 h (in culture media with 10% serum), at the end of incubation cell lysates were prepared and subjected to western blot analysis for EGFR, c-Met, p-ERK, ERK, p-AKT, AKT, c-Myc and Survivin). MDA-MB-231 was included as a positive control. D. Effect of ERK UO-126) and PI-3K (LY294002) inhibitors on growth of MDA-MB-231, MDA-MB-468, BT-549 and BT-20 cells: Cells were plated in six well plates, allowed to attach to the well for 24 h and then treated for 72 h with vehicle only, Deguelin (250 nM), UO126 (10 µM), UO126+Deguelin(250 nM), LY294002 (10 µM) and LY294002+Deguelin (250 nM). The culture media contained 10% serum. Cells were counted at termination. Data represent mean± SE of % of control cells. Number of cells in vehicle treated control was considered as 100%. * indicates significant difference between control and treatment, ** indicates significant difference between two groups shown (p<0.05). Western blot shows changes in p-ERK, ERK, p-AKT, AKT, c-Myc and Survivin levels in MDA-MB-231 cells treated with vehicle, Deguelin (250 nM), UO126 (10 µM) and UO126(10 µM) +Deguelin (250 nM). Representative western pattern for each protein is shown.</p

Schematic diagram showing possible mechanism of Deguelin action in triple negative breast cancer cells.

<p>Deguelin affects EGFR/c-Met and their downstream target molecules such as p-STAT3, p-ERK, p-AKT, c-Myc and Survivin and there by affect growth/survival of breast cancer cells. Three different pathways affected by Deguelin are shown by different colors; STAT3 (green), AKT (blue), ERK (red). Blue arrows with red outline shows molecules shown to be affected by Deguelin in this study. Red dotted line indicates possible alternative IL-6 mediated pathway which is not explored in this study.</p

Effect of Deguelin on the growth of MDA-MB-231, BT-20, BT549 and MDA-MB-468.

<p>The cells were plated in six well plates, incubated at 37°C overnight and then treated with either vehicle only or Deguelin at indicated concentration. Number of cells in each well was counted using coulter counter. Number of cells in vehicle treated well was considered as 100%, data represent mean±SE (% of control) of at least three independent observations. * indicates significant difference between control and treatment.</p