Nitric oxide synthase and breast cancer: role of TIMP-1 in NO-mediated Akt activation

Abstract

Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1) has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Expression of TIMP-1 and CD63 in human breast tumors.

Shown is immunohistochemical staining for TIMP-1 (A–C) and CD63 (D–F). (A) Strong staining of TIMP-1 is shown in the tumor epithelial cells. The surrounding tissue consisting of stromal cells and adipocytes is negative for TIMP-1. (B) Moderate staining of TIMP-1 in tumor epithelial cells is shown in the lower center and to the right. Normal epithelial cells are negative for TIMP-1 (upper left corner). (C) Normal epithelial cells in surrounding non-tumor tissue of an invasive breast tumor are negative for TIMP-1. (D) Strong staining of CD63 in the tumor epithelium. The surrounding tissue consisting of stromal cells and a benign breast duct is mostly negative for CD63. (E) Moderate staining of CD63 in tumor epithelial cells (center). (F) Moderate to strong staining of CD63 in tumor epithelial cells (lower and upper center). Normal epithelial cells to the left and stromal cells are negative for CD63. Magnification: 100× for A, C, D, F; 200× for B and E. Counterstain hematoxylin.

Figure 2. TIMP-1 predicts poor breast cancer survival in patients whose tumors express elevated NOS2 but not in those with low NOS2 expression.

Kaplan-Meier survival analysis demonstrating reduced breast cancer specific survival in A) patients whose tumors express elevated TIMP-1 protein and B) a similar trend when stratified for tumors with high TIMP-1 and high NOS2 protein levels. C) In contrast, TIMP-1 did not predict poor survival of patients with low NOS2 tumor expression.

Figure 3. Transfection of NOS2 expression plamid enhances pAkt.

NOS2 overexpressing MDA-MB-231 breast cancer cells treated with L-Arginine show increased pAkt. Treatment of the same NOS2 transfected cells with the NOS2 inhibitor aminoguanidine (AG) show no increase in pAkt.

Figure 4. Temporal steady state NO flux generated by the NO Donor DETA/NO.

Steady state nM NO levels distributed evenly throughout cell culture media as a function of NO donor (DETA/NO) concentration measured through 24 hr.

Figure 5. Western blots demonstrate the requirement of TIMP-1 for NO-induced Akt phosphorylation in MDA-MB-231 breast cancer cells.

A) Suppression of TIMP-1 protein translation using anti-sense TIMP-1 morpholino through 72 hr. B) TIMP-1 suppression (TIMP-1kd) abolishes NO-induced Akt phosphorylation and reduces the extent of NO-mediated phosphorylation of Akt downstream pro-apoptotic target BAD. The cells were incubated with TIMP-1 morpholino as described in the methods section, serum starved, and then treated with increasing concentrations of DETA/NO for 24 hr. The western blots shown were stripped and reblotted for total Akt are representative of two independent experiments. C–D) Densitometry measurements of pAkt (C) and pBAD (D) relative to total Akt loading control were plotted vs. DETA/NO concentration. Linear regression pAkt/total Akt: control, r2 = 0.974, y-intercept = 90.40+/−10.35, slope = 0.2187+/−0.021 p = 0.0018; TIMP-1 kd, r2 = 0.573, y-intercept = 76.04+/−14.61, slope = 0.0583+/−0.029, p = 0.1381. Linear regression pBAD/total Akt: control, r2 = 0.988, y-intercept = 91.45+/−29.29, slope = 0.9053+/−0.058, p = 0.0006; TIMP-1 kd, r2 = 0.953, y-intercept = 44.60+/−24.30, slope = 0.3747+/−0.048, p = 0.0027.

Figure 6. NO modulation of TIMP-1 protein levels.

A) NO does not significantly effect steady state TIMP-1 mRNA as shown by real time PCR (n = 5). B) NO modulates TIMP-1 protein in the media from MDA-MB-231 breast cancer cells as shown by ELISA assay. The results are presented as mean+/−SEM of n = 4 samples. C) NO modulates TIMP-1 protein in the media from human fibroblasts as shown by western blot.

Figure 7. Confocal image microscopy shows effects of NO on CD63 and TIMP-1 protein co-localization in MDA-MB-231 breast cancer cells treated with DETA/NO.

CD63 (red fluorescence) and TIMP-1 (green fluorescence) in MDA-MB-231 breast cancer cells treated for 24 hr with the NO donor DETA/NO at the following concentrations; A) control, B) 100 µM, C) 500 µM, D) 1000 µM. Panels E and F are the enlarged areas circled in panels C and D, respectively, which show areas of CD63 and TIMP-1 protein co-localization (yellow/orange fluorescence). Cellular nuclei are visualized as blue fluorescence (DAPI). Scale bar = 20 µm for all panels.

Figure 8. Mass spectrum showing nitration of two tyrosine residues (Y95, Y143) in human recombinant TIMP-1 protein treated with 100, 500, or 1000 µM DETA/NO.

A) amino acid sequence of human recombinant TIMP-1 protein (NCBI Reference Sequence: NP_003245.1) and location of tyrosine nitration (Y95, Y143). B) representative mass spectrum of a nitrated peptide from human TIMP-1, illustrating the mass shift of 45 daltons that accompanied nitration.

Figure 9. TIMP-1 immunoprecipitation of DETA/NO treated MDA-MB-231 cell lysates.

A) Western blot showing TIMP-1 protein expression in the lysates of DETA/NO treated MDA-MB-231 breast cancer cells. B) TIMP-1 immunoprecipitated from DETA/NO treated MDA-MB-231 cells and immunoblotted for 3′NT and CD63 demonstrates enhanced TIMP-1 tyrosine nitration and CD63 co-immunoprecipitation associated with DETA/NO exposure.

Figure 10. DETA/NO induced PI3k p85 phosphorylation is suppressed in TIMP-1 knockdown cells.

Figure 11. NOS2/NO promotes TIMP-1-dependent pro-survival signaling through Akt activation in the breast tumor microenvironment.