Amiloride Sensitizes Prostate Cancer Cells to the Reversible Tyrosine Kinase Inhibitor Lapatinib by Modulating ERBB3 Subcellular Localization

Abstract

Neoadjuvant therapy (NAT) has been studied in clinically localized prostate cancer (PCa) to improve the outcomes from radical prostatectomy (RP) by 'debulking' of high-risk PCa; however, using androgen deprivation at this point risks castration resistant PCa (CRPC) clonal proliferation with potentially profound side effects such as fatigue, loss of libido, hot flashes, loss of muscle mass, and weight gain. Our goal is to identify alternative NAT that reduce hormone sensitive PCa (HSPC) without affecting androgen receptor (AR) transcriptional activity. PCa is associated with increased expression and activation of the epidermal growth factor receptor (EGFR) family, including HER2 and ErbB3. Dimerization between these receptors is required for activation of downstream targets involved in tumor progression. The FDA-approved HER2 inhibitor lapatinib has been tested in PCa but was ineffective due to continued activation of ErbB3. We now demonstrate that this is due to ErbB3 being localized to the nucleus in HSPC and thus protected from lapatinib which affect membrane localized HER2/ErbB3 dimers. Here, we show that the well-established, well-tolerated diuretic amiloride hydrochloride dose dependently prevented ErbB3 nuclear localization via formation of plasma membrane localized HER2/ErbB3 dimers. This in turn allowed lapatinib inactivation of these dimers via inhibition of its target HER2, which dephosphorylated downstream survival and proliferation regulators AKT and ERK1/2. Amiloride combined with lapatinib significantly increased apoptosis but did not affect AR transcriptional activity. Thus, our data indicate that a combination of amiloride and lapatinib could target HSPC tumors without problems associated with androgen deprivation therapy in localized PCa.

Keywords: Amiloride; ErbB3; androgen receptor; heregulin-1β; lapatinib; prostate cancer; subcellular localization.

Figure 1. LNCaP cells are sensitive to amiloride and accumulate cytoplasmic ErbB3 upon amiloride treatment.

(A)Hormone-sensitive LNCaP cells were treated with varying concentrations of amiloride for 72h before being lysed, fractionated and analyzed by immunoblot. (B)Immunofluorescence microscopy in LNCaP cells treated with DMSO or 75 μM amiloride for 72 hours (scale bars = 30μm). Note that vehicle treated LNCaP cells expressed nuclear ErbB3 (red) whereas amiloride-treated cells had significantly decreased ErbB3 expression in the nucleus (hollowed out). Location of nuclei are identified by blue DAPI staining. Plasma membrane localization of ErbB3 at cell-cell junction was also noted in amiloride-treated but not in vehicle treated cells. (C) Cells were subjected to viability assays using the stated concentrations of amiloride. Tables shows p-values with respect to DMSO. (D,E) Hormone-insensitive C4–2 or (F,G) the unrelated cell line 22Rv1 cells were also treated with varying concentrations of amiloride for 72h before being subjected to viability assays or lysed and fractionated as previously described. For all viability assays, results were obtained from triplicate experiments. Error bars represent standard deviation. Tables show p-values with respect to DMSO for each tested cell line. All densitometry was performed using ImageJ. C= cytoplasmic and N= nuclear.

Figure 2. Differential activation and dimerization of ErbB family members and their downstream targets in HSPC and CRPC cells with high concentrations of amiloride

(A) HSPC (LNCaP) and CRPC (C4–2, 22Rv1) cells were treated for 72h with 75μM amiloride dissolved in 100% sterile DMSO and stimulated with PBS, EGF or HRG for 15 minutes prior to collection to observe activation of ErbB family members and their downstream targets. Cells were lysed in denaturing lysis buffer before being analysed by immunoblotting. 25μg of protein were loaded per lane. Hsp90 was used as a loading control. (B,C) LNCaP cells (HSPC) were treated with 75μM amiloride or 100% DMSO (0.1% v/v) for 72h and stimulated with PBS, EGF or HRG for 15 minutes to activate ErbB family dimers just prior to collection. 400ug of whole cell lysate were used in each pulldown lane. Mouse IgG antibody was used as an isotype control. Amiloride increases ErbB3-HER2 dimers and stabilizes ErbB3-EGFR dimers. Densitometric analyses was performed with Image J for each cell lines tested. Tables show p-values with respect to DMSO for each tested cell line.

Figure 3. Amiloride efficacy is enhanced by EGFR knockdown in HSPC cells and by HER2 knockdown in CRPC cells.

(A) CRPC C4–2 cells were transfected with control (CT) or EGFR or ErbB3 siRNA or (B) HER2 siRNA and treated with or without 75μM amiloride before being analysed for changes in viability with the MTT assay. Error bars represent standard deviation. Experiments were performed in triplicate. (C) Whole cell immunoblot for siRNA efficacy. 20μg of protein were loaded per lane. Tubulin was used as a loading control. Densitometry was performed using ImageJ. Error bars represent standard error of the mean.

Figure 4. Amiloride enhances the sensitivity of HSPC cells to low concentrations of lapatinib

(A) LNCaP cells were treated with 1–10μM lapatinib and assayed for viability. Lapatinib and amiloride were both dissolved in 100% DMSO. In a head-to-head comparison of lapatinib and amiloride, the combination was additive when cell viability was assayed with the MTT reagent. Experiments were done in triplicate. Error bars represent standard deviation. Table shows p-values with respect to FBS DMSO. (B) Cells were treated with varying concentrations of lapatinib, 10μM amiloride or a combination of the two for 72h before being collected and lysed into cytoplasmic and nuclear fractions as described in earlier figure legends. Co-administration of lapatinib and amiloride increases the accumulation of ErbB3 in the cytoplasmic fraction compared to 2μM lapatinib alone. (C) Cells were treated with 2μM lapatinib, 10μM amiloride or a combination of the two for 72h before being collected, fixed and processed for indirect immunofluorescent microscopy using immunofluorescent-specific antibodies to the C- and N- termini of ErbB3 (‘CTD’ and ‘NTD’ respectively) as previously described. Scale bars = 7.5 μm. (D) LNCaP cells were treated for 72h with lapatinib, amiloride or the combination or 100% sterile DMSO and stimulated with PBS, EGF or HRG for 15 minutes prior to collection to observe activation of ErbB family members and their downstream targets. Cells were lysed in denaturing lysis buffer before being analysed by immunoblotting. 25μg of protein were loaded per lane. Tubulin was used as a loading control.

Figure 5. Amiloride enhances the sensitivity of HSPC and CRPC cell lines to low concentrations of lapatinib.

(A) 22Rv1 cells were treated with varying concentrations of lapatinib, 10μM amiloride or a combination of the two for 72h before being collected and lysed into cytoplasmic and nuclear fractions as described in earlier figure legends. Co-administration of lapatinib and amiloride does not increase the accumulation of ErbB3 in the cytoplasmic fraction. (B) 22Rv1 cells were treated with 1–10μM lapatinib and assayed for viability. Lapatinib was dissolved in 100% DMSO. Experiments were done in triplicate. Error bars represent standard deviation. (C) In a head-to-head comparison of lapatinib and amiloride, the combination was additive when cell viability was assayed with the MTT reagent. Experiments were done in triplicate. Error bars represent standard deviation. Coloured dotted line estimates 50% viability. Table shows p-values with respect to FBS DMSO. (D) 22Rv1 (CRPC) cells were treated for 72h with lapatinib, amiloride or the combination or 100% sterile DMSO and stimulated with PBS, EGF or HRG for 15 minutes prior to collection to observe activation of ErbB family members and their downstream targets. Cells were lysed in denaturing lysis buffer before being analysed by immunoblotting. 25μg of protein were loaded per lane. Tubulin was used as a loading control. Tables show p-values with respect to PBS DMSO in each cell line tested. (E) PC-346C cells were treated with varying concentrations of amiloride and assayed for viability. Amiloride was dissolved in 100% DMSO. Experiments were done in triplicate. Error bars represent standard deviation. (F) PC-346C cells were treated with 1–10μM lapatinib and assayed for viability. Lapatinib was dissolved in 100% DMSO. Experiments were done in triplicate. Error bars represent standard deviation. (G) PC-346C cells were treated with lapatinib, amiloride or the combination, which is shown to be additive when cell viability was assayed with the MTT reagent. Experiments were done in triplicate. Error bars represent standard deviation. Table shows p-values with respect to FBS DMSO. (H) PC-346C cells were treated with varying concentrations of lapatinib, 10μM amiloride or a combination of the two for 72h before being collected and lysed into cytoplasmic and nuclear fractions as described in earlier figure legends.

Figure 6. Amiloride and lapatinib synergize to increase apoptosis in HSPC and CRPC cell lines.

(A-C) HSPC and CRPC cell lines were treated with 100% DMSO, lapatinib, amiloride or the combination (in μM) for 72h before being processed for cell death analysis using annexin V and propidium iodide staining. The percentage of cells undergoing early or late apoptosis with DMSO treatment was set to 100% and values for the various treatment conditions calculated accordingly. Experiments were performed in triplicate. (D) Tables show p-values with respect to DMSO in each cell line tested. Schematic with proposed molecular mechanism of lapatinib-amiloride efficacy. (A) EGFR, HER2 and ErbB3 exist at the cell membrane and signal via pathways such as ERK and AKT. (B,C) ErbB3 monomers cycle between the nucleus and cell membrane. (EGFR and HER2 behave similarly but have been omitted for clarity). (D) Lapatinib is a dual-kinase TKI (tyrosine kinase inhibitor) of HER2 and EGFR dimers but will also inhibit HER2 in HER2-ErbB3 dimers. Lapatinib is unlikely able to inhibit ErbB3 if it is in the nucleus and not at the cell surface. (E) Amiloride is a macropinocytosis inhibitor that prevents internalization of ErbB3 and retains it at the cell surface. As a result, nuclear ErbB3 decreases and cytoplasmic surface ErbB3 increases. (F) Amiloride-induced ErbB3 retention enables its dimerization with HER2, enabling the formation of ErbB3-HER2 dimers which are now inhibited by the addition of low concentrations of lapatinib.