FN14 and GRP94 expression are prognostic/predictive biomarkers of brain metastasis outcome that open up new therapeutic strategies.

Brain metastasis is a devastating problem in patients with breast, lung and melanoma tumors. GRP94 and FN14 are predictive biomarkers over-expressed in primary breast carcinomas that metastasized in brain. To further validate these brain metastasis biomarkers, we performed a multicenter study including 318 patients with breast carcinomas. Among these patients, there were 138 patients with metastasis, of whom 84 had brain metastasis. The likelihood of developing brain metastasis increased by 5.24-fold (95%CI 2.83-9.71) and 2.55- (95%CI 1.52-4.3) in the presence of FN14 and GRP94, respectively. Moreover, FN14 was more sensitive than ErbB2 (38.27 vs. 24.68) with similar specificity (89.43 vs. 89.55) to predict brain metastasis and had identical prognostic value than triple negative patients (p < 0.0001). Furthermore, we used GRP94 and FN14 pathways and GUILD, a network-based disease-gene prioritization program, to pinpoint the genes likely to be therapeutic targets, which resulted in FN14 as the main modulator and thalidomide as the best scored drug. The treatment of mice with brain metastasis improves survival decreasing reactive astrocytes and angiogenesis, and down-regulate FN14 and its ligand TWEAK. In conclusion our results indicate that FN14 and GRP94 are prediction/prognosis markers which open up new possibilities for preventing/treating brain metastasis.


Therapeutic protocols
For brain metastasis treatment, we started therapy on day 14 once the mice had recovered from surgery and after checking the success of cell inoculation.
Lenalidomide (LND) obtained from the Celgene Corporation (Summit, NJ) was injected intraperitoneally in DMSO (Sigma-Aldrich) at 50 mg/Kg/day, every day until the end of the experiment. Taxotere (TXT) and NVP-AUY922 (NVP), both from LC Laboratories, were injected intraperitoneally in DMSO at a dosage of 15 mg/ Kg/day and 30 mg/Kg/day respectively. Docetaxel was administered every 4 days for 2 weeks and NVP every 2 days for 2 weeks.

Histology and immunohistochemical tumor characterization
The morphology of the engrafted tumors was analyzed by H&E staining in paraffin-embedded sections. Determination of FN14 was performed with anti-FN14 at 1/3000 (Santa Cruz Biotechnology, Santa Cruz, CA) (Tecnique) diluted in Dako Real™ Antibody Diluent Buffer (Dakocytomation): Tris buffer, pH 7.2, 15 mM Na 3 N. LSAB+System-HRP (Dakocytomation) was used, including biotinylated anti-rabbit, anti-mouse and anti-goat immunoglobulins in PBS; streptavidin conjugated to HRP in PBS; and liquid 3′3′ diaminobenzidine in chromogen solution.

Western blotting
Cells were lysed in a 1% SDS (v/v) extraction buffer containing an anti-protease cocktail (Roche, Vilvoorde, Belgium). Protein concentrations were determined using the Bradford assay (MicroBCA, Pierce, Belgium). After resolution by SDS-PAGE, electrophoresed proteins were transferred to polyvinylidene fluoride (PVDF) membranes that were blocked and probed with GRP94 (1/1000, Sta Cruz) and the Peroxidase conjugated Antimouse secondary Ab (Pierce, Perbio Science Ltd., Cheshire, U.K). Immunoreactive bands were viewed on a VersaDoc™ (Bio-Rad) Imaging System using the Super Signal west-Pico (Pierce). MWs were established with See Blue Plus2 prestained Standford (Invitrogen, San Diego, CA).

Immune-fluorescence analysis
Cells were analyzed for the expression of FN14. Briefly, 15 × 10 4 or 60 × 10 3 cells were seeded in 6 or 24 well-plates containing coverslips. After 24 hours immunofluorescence was performed anti-FN14 primary antibody in PBS1X and SBF 5%, and then fixed with paraformaldehyde at 4% in PBS 1X for 15 min at 4ºC.
For IF analysis, coverslips were mounted on slides using Vectashield (Vector laboratories) with DAPI, which was used for nucleus visualization. Preparations were analyzed with the Olympus BX60 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) and images were taken and analyzed using a digital camera and Spot 4.2 software (Diagnostic Instruments, Inc., Sterling Heights, MI).

Statistical analysis
To evaluate the correlation between protein expression and brain metastasis, immunostained samples were graded on a three-category scale (negative, weak positive, and strong positive). The marker was classed as being overexpressed in strong positive samples. The association with brain metastasis for each marker was tested using a two-sided Fisher exact test and summarized by calculating the sensitivity among tumors that developed metastasis, and specificity among tumors SUPPLEMENTARY DATA www.impactjournals.com/oncotarget/ Oncotarget, Supplementary Materials 2015 without metastasis, for strong positive values. Positive and negative likelihood ratios were also calculated as integrated predictive indexes, as was the area under the ROC curve. Markers were assessed using a multivariate logistic regression model in a forward stepwise procedure to identify the best combination for predicting brain metastasis. Since ErbB2 was already a known metastasis risk factor, an analysis including ErbB2 as the baseline was also performed, as well as a stratified analysis of each candidate marker within ErbB2-positive and -negative tumors. In all analyses, associations were considered significant when p was less than 0.05.
To compare survival times for the control and LND groups, we used the non-parametric Mann-Whitney test and the log-rank test.
The bioluminescence data were transformed using the log(1 + x) function (where x = AvR), in order to obtain a more regular and positive distribution. Subsequently, these data were normalized by subtracting the first observation (day 14) from each of the following observations. The Student t test was used to compare the treatment groups. Survival curves for each treatment were estimated via the Kaplan-Meier method, and the log-rank test was used to assess the significance of differences.
P-values lower than 0.05 were considered significant.