The cystine/glutamate antiporter system xc- drives breast tumor cell glutamate release and cancer-induced bone pain

Abstract

Bone is one of the leading sites of metastasis for frequently diagnosed malignancies, including those arising in the breast, prostate and lung. Although these cancers develop unnoticed and are painless in their primary sites, bone metastases result in debilitating pain. Deeper investigation of this pain may reveal etiology and lead to early cancer detection. Cancer-induced bone pain (CIBP) is inadequately managed with current standard-of-care analgesics and dramatically diminishes patient quality of life. While CIBP etiology is multifaceted, elevated levels of glutamate, an excitatory neurotransmitter, in the bone-tumor microenvironment may drive maladaptive nociceptive signaling. Here, we establish a relationship between the reactive nitrogen species peroxynitrite, tumor-derived glutamate, and CIBP. In vitro and in a syngeneic in vivo model of breast CIBP, murine mammary adenocarcinoma cells significantly elevated glutamate via the cystine/glutamate antiporter system xc. The well-known system xc inhibitor sulfasalazine significantly reduced levels of glutamate and attenuated CIBP-associated flinching and guarding behaviors. Peroxynitrite, a highly reactive species produced in tumors, significantly increased system xc functional expression and tumor cell glutamate release. Scavenging peroxynitrite with the iron and mangano-based porphyrins, FeTMPyP and SRI10, significantly diminished tumor cell system xc functional expression, reduced femur glutamate levels and mitigated CIBP. In sum, we demonstrate how breast cancer bone metastases upregulate a cystine/glutamate co-transporter to elevate extracellular glutamate. Pharmacological manipulation of peroxynitrite or system xc attenuates CIBP, supporting a role for tumor-derived glutamate in CIBP and validating the targeting of system xc as a novel therapeutic strategy for the management of metastatic bone pain.

Conflict of interest statement

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Figures

Figure 1.

The murine mammary adenocarcinoma cell line 66.1 expresses the cystine/glutamate antiporter system xc−. (A) Representative images of 66.1 XCT and DAPI staining, as indicated. 66.1 cell glutamate release was quantified over a 24-hour period in untreated cells (B), and at 18 hours in those exposed to vehicle, sulfasalazine (SSZ) (C) or (S)-4-carboxyphenylglycine (CPG-4) (D). Asterisks denote values statistically different from time 0 or vehicle control group, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

Blockade of system xc− with sulfasalazine (SSZ) attenuates cancer-induced bone pain behaviors and reduces femur glutamate. Animal femurs were injected with breast cancer cells (66.1) or cell-free media (sham) after baseline (presurgery) behavioral measurements. SSZ (30 mg/kg, i.p.) or vehicle (10 mL/kg, i.p.) was administered after behavioral measurements on postsurgery day 7 and continued for 3 days (q.d.). Cancer-induced spontaneous flinching (A) and guarding (B) were significantly reduced 60 minutes after SSZ treatment on day 10, as compared with vehicle-treated animals. Femur contents from inoculated femur (ipsilateral) (C) and contralateral femur (D) were collected on day 10 for glutamate analysis; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

Peroxynitrite regulates system xc− functional expression. 66.1 cells were treated SIN-1 (50 nm–5 μM) or vehicle. Eighteen hours after treatment, cells were harvested and analyzed by Western blot analysis for XCT (A and B). SIN-1 stimulated 66.1 glutamate release in the absence (C) or presence (D) of sulfasalazine was quantified. 66.1 cells were exposed to SIN-1 in the presence of FeTMPyP or vehicle. Eighteen hours after treatment, cells were harvested and analyzed through Western blot analysis for expression of XCT (E and F). 66.1 glutamate release in the presence of SIN-1 and FeTMPyP, SRI10, or vehicle was quantified (G and H). Asterisks represent data points that are significantly different from those of vehicle control, unless otherwise indicated; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

Repeated FeTMPyP treatment modulates spontaneous cancer-induced bone pain behaviors and system xc− functional expression in vivo. Animal femurs were injected with breast cancer cells (66.1) or cell-free media (sham) after baseline (presurgery) behavioral measurements. On day 7 after femoral inoculation, 66.1 animals demonstrated bone cancer–induced flinching (A) and guarding (B). FeTMPyP (10 mg/kg, i.p.) or vehicle (saline, 10 mL/kg, i.p.) was administered after behavioral measurements day 7 and continued to day 14 (q.d.). Spontaneous flinching and guarding were assessed before and after treatment on days 10 and 14 (A and B). Femur marrow from inoculated femur (ipsilateral) (C) and contralateral femur (D) was collected from tumor-bearing animals before or after the final treatment on day 14 for glutamate analysis. Day 14 ipsilateral marrow samples from tumor-bearing animals were analyzed for XCT expression using Western blot analysis (E–H); *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Repeated FeTMPyP does not alter tumor burden. Animal femurs were inoculated with cell-free media (sham) or breast cancer cells (66.1). FeTMPyP (10 mg/kg, i.p., q.d.) or vehicle (saline, 10 mL/kg, i.p., q.d.) was administered on postsurgery days 7 to 14. On day 14, femurs were collected, paraffin-embedded, and stained with hematoxylin and eosin (H&E) to visualize normal marrow elements and cancer cells under bright field microscopy. Representative images (A) and quantification of cancer cell marrow occupancy (B) are shown.

Figure 6.

Repeated FeTMPyP does not alter bone integrity. Animal femurs were inoculated with cell-free media (sham) or breast cancer cells (66.1). FeTMPyP (10 mg/kg, i.p., q.d.) or vehicle (saline, 10 mL/kg, i.p., q.d.) was administered on postsurgery days 7 to 14. On day 14, live radiographs were taken. Bone loss was rated by 3 blinded observers according to an established scale (A). Elevated bone loss scores were found in the tumor-bearing animals as compared with sham controls, but no treatment differences were evident (B); ***P < 0.001.

Figure 7.

Repeated SRI10 treatment attenuates spontaneous cancer-induced bone pain behaviors and reduces femur glutamate. Animal femurs were injected with breast cancer cells (66.1) or cell-free media (sham) after baseline (presurgery) behavioral measurements. SRI10 (3 mg/kg, i.p.) or vehicle (10 mL/kg, i.p.) was administered after behavioral measurements day 7 and continued to day 14 (q.d.). Spontaneous flinching (A) and guarding (B) were assessed before and after treatment on days 10 and 14. Femur marrow from inoculated femur (ipsilateral) (C) and contralateral femur (D) was collected from tumor-bearing animals before or after the final treatment on day 14 for glutamate analysis; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8.

System xc− can be targeted directly or indirectly to reduce glutamate in the bone-tumor microenvironment and assuage cancer-induced bone pain (CIBP). System xc− can be targeted pharmacologically through direct blockage with sulfasalazine or through elimination of peroxynitrite with the decomposition catalysts FeTMPyP and SRI10. Both therapeutic strategies reduced glutamate in the bone–tumor microenvironment and CIBP-related behaviors.