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BackgroundAround 15–20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance.MethodsRANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling.ResultsRANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status.ConclusionsOur data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated.

Additional file 5: Figure S5. A. Relative number of living (relative survival) SKBR3 and SKLR control cells incubated for 4 days with the indicated concentrations of lapatinib and stimulated with RANKL. Cells were seeded in growth media, 100 ng/ml RANKL were added 24h after seeding, lapatinib was added 24 h later and cells were analyzed with CCK8 as detailed in methods. Determinations were done in triplicates, mean values are depicted from n = 5 independent experiments and SD and p-value (**) calculated by one-way ANOVA is depicted (p ≤ 0.05 for SKBR3 vs SKLR and SKLR +RANKL, SKBR3 +RANKL vs SKLR and SKLR +RANKL; n.s. for SKBR3 vs SKBR3 +RANKL and SKLR vs SKLR +RANKL). Significance of relative survival was calculated for each concentration using two-tailed p values for one sample t test. RANKL significantly increased survival of SKLR cells at 0.018 μM of lapatinib (p = 0.019). B. Western blot showing the total levels of IκB, p65, ERK1/2, AKT and HER2 in SKBR3 control, SKLR control and SKLR sh#3 cells treated with RANKL or lapatinib as depicted in Fig. 4c. Cells were serum starved for 12 h and then treated with lapatinib (2 h) or RANKL (10 min) before processing them. Tubulin was used as a loading control. C. Table depicting the relative phospho-protein levels of the indicated proteins from the western blots shown in Fig. 4c and Fig. S5B determined by densitometry analyses with Image J.

Additional file 7: Figure S7. Western blot analyses of HER2 (p-HER2, p-ERK1/2) and NF-κB (p-p65) pathway activation in SKBR3, BT474 and HCC1954 cells stably transduced with empty or RANK overexpressing (RANK) vectors. Cells were cultured in media without FBS for 12 h, followed by stimulation with EGF (100 ng/ml) (upper panels) or heregulin (HRG 10 ng/ml) (lower panels) for the indicated times. Tubulin was used as a loading control.

Additional file 6: Figure S6. A. Relative number of living (relative survival) SKBR3 RANK cells stimulated with RANKL in the presence or absence of denosumab (DNS) and incubated for 4 days with the indicated concentrations of lapatinib. Cells were seeded in growth media with/without denosumab (1 μg/ml), lapatinib was added after 24 h stimulation with 100 ng/ml RANKL, and cell viability was analyzed with CCK8 as detailed in methods. Determinations were done in triplicates, mean values from n ≥ 2 independent experiments and SD are depicted. B. Western blot analyses of total levels of p65, ERK1/2 and HER2 in whole lysates from SKBR3, BT474 and HCC1954 cells stably transduced with control (empty) or RANK overexpressing (RANK) vectors as depicted in Fig. 5d. Before collecting the cells, they were cultured in media without FBS for 12 h, pretreated with/without lapatinib for 2 h followed by 10 min stimulation with RANKL. Tubulin was used as a loading control. C. Table depicting the relative phospho-protein levels of the indicated proteins from the western blots shown in Fig. 5d and Fig. S6B determined by densitometry analyses with Image J.

Additional file 3: Figure S3. RANK and RANKL expression in breast cancer samples from the PAMELA clinical trial. A. Expression of RANK and RANKL across the intrinsic molecular subtypes from the PAMELA study. P values were calculated by comparing mean values across all groups. B. Scatter plots of RANK and RANKL expression versus ERBB2 expression for baseline samples in the PAMELA study. Solid line in each figure represents the regression line. Pearson correlation coefficient (r) with significance (p value) is presented in each figure. C. Pearson correlation between single genes and gene expression signatures evaluated in baseline samples from the PAMELA study.

Additional file 4: Figure S4. RANK but not RANKL expression increased after dual anti-HER2 therapy in HR+ and HR- patient samples (n = 151) from the PAMELA trial. A and B. Ladder plots (left panels) showing RANK and RANKL gene expression in PAMELA HER2-positive HR+ (A) and HR- (B) tumors before (baseline) and after (surgery) dual anti-HER2 treatment. An increase in gene expression is represented in red and a decrease in blue. Each line represents a tumor sample from one patient. P values in A were calculated by comparing mean values between both groups and in B were determined by paired two-tailed t-tests. Density plots (right panels) showing RANK and RANKL gene expression in PAMELA HER2-positive HR+ (A) and HR- (B) tumors before (baseline) and after (surgery) treatment.

Additional file 2: Figure S2. RANK and RANKL staining in TMAs. A. Pictures of RANK and RANKL protein expression analyzed by IHC in the TMA cores from the treatment-naïve cohort. B. Pictures of RANK and RANKL protein expression analyzed by IHC in the TMA cores from the anti-HER2 resistant cohort.

Additional file 2: Figure S2. RANK and RANKL staining in TMAs. A. Pictures of RANK and RANKL protein expression analyzed by IHC in the TMA cores from the treatment-naïve cohort. B. Pictures of RANK and RANKL protein expression analyzed by IHC in the TMA cores from the anti-HER2 resistant cohort.

Additional file 4: Figure S4. RANK but not RANKL expression increased after dual anti-HER2 therapy in HR+ and HR- patient samples (n = 151) from the PAMELA trial. A and B. Ladder plots (left panels) showing RANK and RANKL gene expression in PAMELA HER2-positive HR+ (A) and HR- (B) tumors before (baseline) and after (surgery) dual anti-HER2 treatment. An increase in gene expression is represented in red and a decrease in blue. Each line represents a tumor sample from one patient. P values in A were calculated by comparing mean values between both groups and in B were determined by paired two-tailed t-tests. Density plots (right panels) showing RANK and RANKL gene expression in PAMELA HER2-positive HR+ (A) and HR- (B) tumors before (baseline) and after (surgery) treatment.

Additional file 1: Figure S1. TMA H-scores and controls. A. RANK and RANKL H-scores in HER2-positive breast cancer samples, treatment-naïve (left panels) or anti-HER2-resistant (right panels). In treatment-naïve TMAs, each number represents a “core” from a single patient. In anti-HER2-resistant TMAs, scored independent tumor cores are numbered for each patient (after the symbol #). B. Representative pictures of human breast tumors from patient-derived xenografts used as positive and negative controls for RANK and RANKL IHC.

Additional file 1: Figure S1. TMA H-scores and controls. A. RANK and RANKL H-scores in HER2-positive breast cancer samples, treatment-naïve (left panels) or anti-HER2-resistant (right panels). In treatment-naïve TMAs, each number represents a “core” from a single patient. In anti-HER2-resistant TMAs, scored independent tumor cores are numbered for each patient (after the symbol #). B. Representative pictures of human breast tumors from patient-derived xenografts used as positive and negative controls for RANK and RANKL IHC.