Adrenergic modulation of focal adhesion kinase protects human ovarian cancer cells from anoikis

Abstract

Chronic stress is associated with hormonal changes that are known to affect multiple systems, including the immune and endocrine systems, but the effects of stress on cancer growth and progression are not fully understood. Here, we demonstrate that human ovarian cancer cells exposed to either norepinephrine or epinephrine exhibit lower levels of anoikis, the process by which cells enter apoptosis when separated from ECM and neighboring cells. In an orthotopic mouse model of human ovarian cancer, restraint stress and the associated increases in norepinephrine and epinephrine protected the tumor cells from anoikis and promoted their growth by activating focal adhesion kinase (FAK). These effects involved phosphorylation of FAKY397, which was itself associated with actin-dependent Src interaction with membrane-associated FAK. Importantly, in human ovarian cancer patients, behavioral states related to greater adrenergic activity were associated with higher levels of pFAKY397, which was in turn linked to substantially accelerated mortality. These data suggest that FAK modulation by stress hormones, especially norepinephrine and epinephrine, can contribute to tumor progression in patients with ovarian cancer and may point to potential new therapeutic targets for cancer management.

Figure 1. Effect of (A) 10 μM norepinephrine or (B) 10 μM epinephrine on anoikis in ovarian cancer cells.

Gray bars, vehicle treatment; black bars, norepinephrine (A) or epinephrine (B) treatment. Results represent the mean ± SEM. *P < 0.05.

Figure 2. Effect of catecholamines on FAK activation.

(A) SKOV3ip1 or EG cells were plated and treated with either norepinephrine (NE) or epinephrine (Epi) and then subjected to Western blot analysis for FAK or pFAK^Y397. (B) SKOV3ip1 cells kept in suspension were treated with norepinephrine, followed by Western blot for FAK and pFAK^Y397. (C) SKOV3ip1 cells were treated with norepinephrine, then subjected to immunofluorescence analysis for pFAK^Y397 and actin. Quantification of pFAK^Y397 staining is shown in the graph. Scale bars: 10 μm. (D) Effect of 10 μM norepinephrine with or without FAK silencing with siRNA on anoikis. Results represent the mean ± SEM. *P ≤ 0.01.

Figure 3. Effect of propranolol (nonspecific β-antagonist), atenolol (β₁ antagonist), butoxamine (β₂ antagonist), or SR59230A (β₃-antagonist) on FAK and pFAK^Y397 in (A) SKOV3ip1 and (B) EG cells.

(C) Effect of 10 μM norepinephrine with or without propranolol or ADRB1- or ADRB2-targeted (β1 or β2) siRNA in SKOV3ip1 cells on anoikis. (D) Effect of β1 and β2 targeted siRNA on pFAK^Y397 and FAK in SKOV3ip1 cells. In A, B, and D, the immunoblot is shown at the top, and quantification of band intensity relative to total FAK intensity is shown below. For all panels, results represent the mean ± SEM of triplicate experiments. *P < 0.01.

Figure 4. Mechanism of norepinephrine-mediated (NE-mediated) FAK activation.

(A) SKOV3ip1 cells stimulated with 10 μM norepinephrine were treated with either the Src inhibitor PP2 or its inactive counterpart PP3 followed by immunoblotting for FAK and pFAK^Y397. In addition, the effect of Src silencing with siRNA was examined on norepinephrine-mediated FAK activation. (B) SYF-null cells transfected with either empty vector (EV) or Src (WT Src) were stimulated with 10 μM norepinephrine, followed by immunoblotting for FAK and pFAK^Y397. (C) SKOV3ip1 cells treated with 10 μM norepinephrine were subjected to immunoprecipitation for Src (left) or FAK (right), followed by immunoblotting for FAK and Src. (D) Cells stimulated with 10 μM norepinephrine were treated with thapsigargin followed by Western blot for pFAK^Y397 and FAK (left). Cells stimulated with 10 μM norepinephrine were treated with EGTA followed by immunoprecipitation for FAK, then immunoblotting for FAK and Src (right). (E) SKOV3ip1 cells stimulated with 10 μM norepinephrine were treated with cytochalasin D (Cyt D), followed by Western blot for pFAK^Y397 and FAK. In A, D, and E, the immunoblot is shown at the top, and quantification of band intensity relative to total FAK intensity is shown below.

Figure 5. Effect of (A) chronic stress or (B) isoproterenol on tumor cell apoptosis in ascites as a reflection of in vivo anoikis using the 2774 model.

Effects of control siRNA-DOPC or FAK siRNA-DOPC on stress-induced in vivo (C) SKOV3ip1 or (D) HeyA8 tumor growth. (E) Effect of stress on tumor cell apoptosis in the SKOV3ip1 model. (F) Effect of propranolol or FAK siRNA-DOPC on tumor cell apoptosis in ascites as a measure of anoikis using the 2774 model. Results represent the mean ± SEM; n = 10 mice per group. *P < 0.01.

Figure 6. Clinical significance of FAK activation in ovarian carcinoma.

(A) Representative images of human ovarian tumors with low or high immunohistochemical staining for FAK and pFAK^Y397. Original magnification, ×200. (B) Kaplan-Meier curves of disease-specific mortality for patients with epithelial ovarian carcinoma based on FAK or pFAK^Y397 expression. The log-rank test (2-sided) was used to compare differences between groups. Percentage of ovarian cancers with high FAK or pFAK^Y397 expression based on CESD scores of at least 16 (C) or tumoral norepinephrine (NE) levels (greater versus less than median value of 0.84 pg/mg) (D).