Role of RPL39 in Metaplastic Breast Cancer

Abstract

Background: Metaplastic breast cancer is one of the most therapeutically challenging forms of breast cancer because of its highly heterogeneous and chemoresistant nature. We have previously demonstrated that ribosomal protein L39 (RPL39) and its gain-of-function mutation A14V have oncogenic activity in triple-negative breast cancer and this activity may be mediated through inducible nitric oxide synthase (iNOS). The function of RPL39 and A14V in other breast cancer subtypes is currently unknown. The objective of this study was to determine the role and mechanism of action of RPL39 in metaplastic breast cancer.

Methods: Both competitive allele-specific and droplet digital polymerase chain reaction were used to determine the RPL39 A14V mutation rate in metaplastic breast cancer patient samples. The impact of RPL39 and iNOS expression on patient overall survival was estimated using the Kaplan-Meier method. Co-immunoprecipitation and immunoblot analyses were used for mechanistic evaluation of RPL39.

Results: The RPL39 A14V mutation rate was 97.5% (39/40 tumor samples). High RPL39 (hazard ratio = 0.71, 95% confidence interval = 0.55 to 0.91, P = 006) and iNOS expression (P = 003) were associated with reduced patient overall survival. iNOS inhibition with the pan-NOS inhibitor NG-methyl-L-arginine acetate decreased in vitro proliferation and migration, in vivo tumor growth in both BCM-4664 and BCM-3807 patient-derived xenograft models (P = 04 and P = 02, respectively), and in vitro and in vivo chemoresistance. Mechanistically, RPL39 mediated its cancer-promoting actions through iNOS signaling, which was driven by the RNA editing enzyme adenosine deaminase acting on RNA 1.

Conclusion: NOS inhibitors and RNA editing modulators may offer novel treatment options for metaplastic breast cancer.

© The Author 2016. Published by Oxford University Press.

Figures

Figure 1.

RPL39 (A14V) mutation status analyzed in metaplastic breast cancer. A) The relationship between ribosomal protein L39 (RPL39) expression and overall survival was assessed among 457 triple-negative breast cancer (TNBC) patients. The P value was calculated using a log-rank test.B)RPL39 A14V mutation rate was determined by competitive allele-specific TaqMan (CAST) polymerase chain reaction (PCR) analysis of tumor samples from patients with nonmetaplastic and metaplastic breast cancer (each n = 40). Fisher’s exact test was used to compare the mutation rate in metaplastic breast cancer vs nonmetaplastic breast cancer. PIK3CAmutations (H1047R and E545K) rates are also shown. C) The results of the CAST PCR analysis were confirmed by droplet digital PCR. Wilcoxon rank-sum test was used to determine statistically significant differences in the fractional abundance of the RPL39 A14V mutation between metaplastic breast cancer and TNBC. We used a threshold of 0.1% fractional abundance to define low RPL39 expression level. All statistical tests were two-sided. CI = confidence interval; HR = hazard ratio; MBC = metaplastic breast cancer; TNBC = triple-negative breast cancer.

Figure 2.

Correlation between inducible nitric oxide synthase (iNOS) expression and patient overall survival in metaplastic breast cancer. iNOS expression levels were evaluated by immunohistochemical analysis in a cohort of metaplastic breast cancer patient tumor samples (n = 40). The graph presents Kaplan-Meier estimated survival curves for the high and low iNOS expression groups. A two-sided log-rank test was used to determine the statistical significance of the observed difference in survival. iNOS = inducible nitric oxide synthase.

Figure 3.

Role of inducible nitric oxide synthase (iNOS) inhibition in metaplastic breast cancer cell proliferation and migration in vitro. A) Metaplastic breast cancer cell lines HS578T and BT549 were treated with vehicle control or the indicated concentrations of L-NMMA for 72 hours. Proliferation was assessed by WST1 assay (HS578T: mean = 0.76, SD = 0.03, P = .04; BT549: mean = 0.74, SD = 0.01, P = .04). B) Migration by scratch assay was determined in HS578T and BT549 cells treated with vehicle control or 1 mM L-NMMA for 72 hours. (HS578T: mean = 12, SD = 1.5,P = .02; BT549: mean = 24, SD = 3.4, P = .02).C) HS578T and BT549 cells were treated with vehicle control, L-NMMA alone (1 mM), docetaxel alone (1 nM), or combined docetaxel and L-NMMA for 72 hours and the number of apoptotic cells determined by Annexin V staining (docetaxel vs combination therapy, HS578T, mean = 23.8, SD = 4.1, P = .03; BT549 mean = 22.1, SD = 1.8, P = .03). Data presented are based on three independent experiments, and error bars represent the standard deviation. P values were calculated using one-way analysis of variance, followed by the Tukey test for pairwise comparisons. All statistical tests were two-sided.

Figure 4.

Testing the impact of inducible nitric oxide synthase (iNOS) inhibition on in vivo tumor growth and chemoresistance in metaplastic breast cancer. A) The effect of iNOS inhibition on in vivo tumor growth was determined using two metaplastic breast cancer patient-derived xenograft models, BCM-4664 and BCM-3807. Mice (n = 10 per group) were treated with vehicle (saline, intraperitoneal [i.p.], daily) or L-NMMA (200 mg/kg, i.p., daily) for 28 days. Tumor volume was measured twice weekly. P values were calculated using Student’st test. Data are presented as the mean, with the error bars representing 95% confidence intervals (1.96SEM). B)Target engagement was verified by immunohistochemical analysis of iNOS expression in L-NMMA-treated BCM-4664 and BCM-3807 tumors. C) BCM-4664 mice were treated with docetaxel alone (20 mg/kg, i.p., once every 14 days) or in combination with L-NMMA for 42 days. Tumor volume was measured twice weekly. Data were analyzed using Student’s t test and presented as the mean ± 95% confidence interval (1.96 SEM). All statistical tests were two-sided.

Figure 5.

Signal transduction pathway of RPL39 in metaplastic breast cancer. A)Ingenuity pathway analysis was done to identify potential links between RPL39 and iNOS. B) Immunoblot analysis of ADAR1 and phosphorylated STAT3 was performed in HS578T and BT549 cells transfected with a plasmid to overexpress RPL39 or RPL39 A14V. C) Ubiquitin C (UBC) was immunoprecipitated from HS578T and BT549 cell extracts. Immunoprecipitates were immunoblotted to detect the presence of the indicated proteins. D)ADAR1 was immunoprecipitated from HS578T and BT549 cell extracts. Immunoprecipitates were immunoblotted to detect the presence of the indicated proteins. E)Immunoblot analysis was performed to determine the expression of UBC, iNOS, and RPL39 in RPL39-overexpressing and siRNA-treated HS578T and BT549 cells.F) Immunoblot analysis was performed to determine the expression of iNOS, ADAR1, and phosphorylated STAT3 in iNOS and RPL39 in RPL39-overexpressing HS578T and BT549 cells treated with RPL39 or iNOS siRNA. G) Schematic of the proposed RPL39/UBC/ADAR1/iNOS/STAT3 signaling pathway is shown. β-actin served as a loading control in (B–F). ADAR1 = adenosine deaminase acting on RNA 1; IB = immunoblot; iNOS = inducible nitric oxide synthase; IP = immunoprecipitate; p = phosphorylated; RPL39 = ribosomal protein L39; STAT3 = signal transducer and activator of transcription 3; UBC = ubiquitin C.

Figure 6.

Role of RPL39 and RNA editing in metaplastic breast cancer cell proliferation and migration. A and B) RNA editing analysis is shown forABOBEC3D, GLI1, and AZIN1 in RPL39-overexpressing HS578T and BT549 cells (HS578T cells:ABOBEC3D, mean = 16.1, SD = 2.8, P = .03;GLI1, mean = 17.2, SD = 3.1, P = .04;AZIN1, mean = 21.4, SD = 1.8, P = .02) (BT549 cells: ABOBEC3D, mean = 2.1, SD = 0.3, P = .02;GLI1, mean = 1.6, SD = 0.2, P = .04;AZIN1, mean = 1.9, SD = 0.2, P = .04).C) Immunoblot analysis with the indicated antibodies was performed using cell lysates from RPL39-overexpressing HS578T and BT549 cells treated with ADAR1-specific siRNA. D) Migration (P < .001) and(E) proliferation indices were measured in HS578T control cells, RPL39-overexpressing cells, and RPL39-overexpressing cells treated with ADAR1-specific siRNA (P < .001). Error barsrepresent standard deviation from the mean. Two-sided Student’s ttest was used to calculate the P values. ADAR1 = adenosine deaminase acting on RNA 1; APOBEC3D = catalytic polypeptide-like 3D; AZIN1 = antizyme inhibitor 1; GLI1 = GLI family zinc finger 1; iNOS = inducible nitric oxide synthase; MDM2 = mouse double minute 2; RPL39 = ribosomal protein L39; UBC = ubiquitin C.