β-endorphin at the intersection of pain and cancer progression: Preclinical evidence

Abstract

We examined the association between endogenous opioid β-endorphin, cancer progression and pain in a transgenic mouse model of breast cancer, with a rat C3(1) simian virus 40 large tumor antigen fusion gene (C3TAg). C3TAg mice develop ductal epithelial atypia at 8 weeks, progression to intra-epithelial neoplasia at 12 weeks, and invasive carcinoma with palpable tumors at 16 weeks. Consistent with invasive carcinoma at 4 months of age, C3TAg mice demonstrate a significant increase in hyperalgesia compared to younger C3TAg or control FVBN mice without tumors. Our data show that the growing tumor contributes to circulating β-endorphin. As an endogenous ligand of mu opioid receptor, β-endorphin has analgesic activity. Paradoxically, we observed an increase in pain in transgenic breast cancer mice with significantly high circulating and tumor-associated β-endorphin. Increased circulating β-endorphin correlates with increasing tumor burden. β-endorphin induced the activation of mitogenic and survival-promoting signaling pathways, MAPK/ERK 1/2, STAT3 and Akt, observed by us in human MDA-MB-231 cells suggesting a role for β-endorphin in breast cancer progression and associated pain.

Keywords: Breast cancer; Morphine; Opioid; Pain; β-Endorphin.

Figure 1:. Hyperalgesia increases with age in transgenic mice with breast cancer.

(A) Paw withdrawal frequency (PWF) in response to von Frey filament to test mechanical hyperalgesia. Higher PWF demonstrates more hyperalgesia. (B) Grip force to test musculoskeletal/deep tissue hyperalgesia. Lower grip force demonstrates more hyperalgesia. (C) Paw withdrawal latency (PWL) in response to a radiant heat stimulus applied to the plantar surface of the hind paw to test heat hyperalgesia. Lower PWL demonstrates more hyperalgesia. (D) Paw withdrawal frequency (PWF) over a period of 2 minutes when placed on a cold plate at 4°C. Higher PWF demonstrates more hyperalgesia. (E) Body weight of mice used for hyperalgesia measurements. Data are shown as mean ± SEM. Statistical analysis: One-way ANOVA/Bonferroni’s post-hoc analysis. A-D, ** P<0.0001 vs. 1.0-month old C3TAg; $$ P<0 .0001 vs. 2.1 months old C3TAg, ## P<0.0001 vs. 5.2 months old FVBN. FVBN, wild type genetic background mice and C3TAg, transgenic mice with breast cancer.

Figure 2:. Constitutive increase in circulating β-endorphin with increasing tumor burden in transgenic mice with breast cancer.

(A) β-endorphin was analyzed in the plasma from C3TAg mice without tumors at 1.8 months and 5.2 month old with tumors and from control FVBN mice at 7.8 months of age. Plasma β-endorphin is significantly higher in 5.2 month old C3TAg mice with tumors compared to 1.8 month old C3TAg mice or FVBN mice without tumors. Notably, no significant difference in plasma β-endorphin concentration was observed between FVBN and 1.8 month old C3TAg mice. (B) Plasma and tumors were obtained from C3TAg mice at 5.2 months of age. Each value represents the plasma β-endorphin and correlative tumor weight from each mouse. Plasma β-endorphin increases with increasing tumor weight in C3TAg mice. Statistical analysis: (A) One-way ANOVA/Bonferroni’s post-hoc analysis; (B) Pearson’s correlation.

Figure 3:. Increased β-endorphin expression in tumors of older transgenic breast cancer mice compared to control breast tissue from younger mice.

Breast tumors from ~6 month old C3TAg mice and non-tumor breast tissue from ~3 month old C3TAg mice were analyzed for β-endorphin expression. Significantly higher β-endorphin concentration was observed in the tumors compared to non-cancerous mammary tissue. Data are shown as mean ± SEM. Statistical analysis: Student’s two-tailed t-test.

Figure 4:. β-endorphin stimulates mitogenic and survival promoting signaling in human MDA-MB-231 breast cancer cells.

In vitro human MDA-MB-231 breast cancer cells were incubated with 1 nM β-endorphin for indicated time or with vehicle. Compared to total protein, phosphorylated signaling molecules are shown at 5, 30 or 60 minutes after β-endorphin stimulation: (A) Phospho- to total-MAPK/ERK1/2, (B) Phospho- to total-STAT3 and, (C) Phospho- to total-Akt. Each image represents 3 separate and reproducible experiments. Data are shown as mean ± SEM. Statistical analysis: Student’s two-tailed t-test. * P<0.05 and *** P<0.001, compared to vehicle.