Enhancing the effectiveness of androgen deprivation in prostate cancer by inducing Filamin A nuclear localization

Abstract

As prostate cancer (CaP) is regulated by androgen receptor (AR) activity, metastatic CaP is treated with androgen deprivation therapy (ADT). Despite initial response, patients on ADT eventually progress to castration-resistant CaP (CRPC), which is currently incurable. We previously showed that cleavage of the 280 kDa structural protein Filamin A (FlnA) to a 90 kDa fragment, and nuclear localization of the cleaved product, sensitized CRPC cells to ADT. Hence, treatment promoting FlnA nuclear localization would enhance androgen responsiveness. Here, we show that FlnA nuclear localization induced apoptosis in CRPC cells during ADT, identifying it as a treatment tool in advanced CaP. Significantly, the natural product genistein combined polysaccharide (GCP) had a similar effect. Investigation of the mechanism of GCP-induced apoptosis showed that GCP induced FlnA cleavage and nuclear localization and that apoptosis resulting from GCP treatment was mediated by FlnA nuclear localization. Two main components of GCP are genistein and daidzein: the ability of GCP to induce G2 arrest was due to genistein whereas sensitivity to ADT stemmed from daidzein; hence, both were needed to mediate GCP's effects. FlnA cleavage is regulated by its phosphorylation; we show that ADT enhanced FlnA phosphorylation, which prevented its cleavage, whereas GCP inhibited FlnA phosphorylation, thereby sensitizing CaP cells to ADT. In a mouse model of CaP recurrence, GCP, but not vehicle, impeded relapse following castration, indicating that GCP, when administered with ADT, interrupted the development of CRPC. These results demonstrate the efficacy of GCP in promoting FlnA nuclear localization and enhancing androgen responsiveness in CaP.

Figure 1. Nuclear localization of a 90 kDa FlnA fragment induces apoptosis in prostate cancer cells

(A) (left) LNCaP and C4-2 cells were treated with increasing doses of the anti-androgen bicalutamide for 48 hrs and the percentage of cells in S-phase was analyzed by flow cytometry in PI-stained cells. (right) Untreated LNCaP and C4-2 cells. Western blotting revealed that both cell lines expressed equal levels of 280 kDa FlnA, but LNCaP cells expressed higher levels of 90 kDa FlnA compared to C4-2. (B) LNCaP, C4-2 and C4-2 cells stably transfected with FlnA 16-24 (C4-2 16-24) were stained by immunofluorescence for FlnA with an antibody specific to the C-terminus of FlnA. Immunofluorescent staining shows nuclear localization of 90kDa FlnA in LNCaP cells whereas in C4-2 cells FlnA was mainly localized to the cytoplasm but not in the nucleoplasm, although bright staining in the nucleolus was also detected. In contrast, C4-2 16-24 cells expressed this protein equally in the cytoplasm and the nucleus, while nucleolar staining could still be detected. Scale bar: 10 μm. (C) Subcellular fractionation of CWR22Rv1 cells transfected with 1 μg/ml cDNA expressing FlnA 16-24 showing that the 16-24 fragment goes into the nucleus. Some full length FlnA enters the nucleus when transfected with FlnA 16-24 due to nucleolar localization. (D) Flow cytometric analysis in PI-stained, ethanol fixed C4-2 cells to determine the % cells undergoing apoptosis. Apoptosis was considered to be the cells staining with Annexin V, and showed that transfection with FlnA 16-24 induced apoptosis in C4-2 cells.

Figure 2. Treatment with genistein combined polysaccharide (GCP) replicates the effects of FlnA nuclear localization

(A) Expression of the transfected proteins when CWR22Rv1 cells were transiently transfected for 48 hours with 2 μg/ml of an empty vector, full-length FlnA (280 kDa), FlnA 1-15 (170 kDa) or FlnA 16-24 (90 kDa). Actin and Lamin A were used as markers of the cytoplasm and the nucleus, respectively. (B) As estimated by MTT assay, neither 10 μM bicalutamide nor FlnA 16-24 individually had significant effects on the growth of CWR22Rv1 cells, but in combination, they prevented cell growth. Each point on the graph represents mean ± S.D. of 3 independent readings. (C) CWR22Rv1 cells underwent apoptosis when transfected with FlnA 16-24 but not when transfected with an empty vector, FlnA 280 or FlnA 1-15 as determined by flow cytometry after 48 hours in Annexin V and propidium iodide stained cells. (D) (upper) MTT assay showing effect of CWR22Rv1 cells with GCP or vehicle in FBS and CSS containing medium. Each point on the graph represents mean ± S.D. of 3 independent readings. (lower) Analysis of apoptosis by flow cytometry in propidium iodide and Annexin V stained cells demonstrating that CWR22Rv1 cells treated with 100 μg/ml GCP for 48 hours experienced more apoptosis than vehicle treated cells. Early apoptosis: Annexin V staining, Mid-apoptosis: Annexin V+PI staining, Late apoptosis: PI staining. Results presented represent numbers after subtraction from untreated cells. Note the increase in apoptosis in cells cultured in CSS vs FBS containing medium. (E) Western blots demonstrating that 100 μg/ml GCP after 48 hrs increased apoptosis as demonstrated by cleaved Caspase 3 and PARP levels.

Figure 3. Treatment with GCP induces FlnA cleavage to the 90 kDa fragment which mediates GCP-induced apoptosis

(A) Luciferase assay to determine AR transcriptional activity on a PSA promoter to determine the effect of 10 μM bicalutamide or vehicle in CWR-R1 cells. Cells were cultured in FBS and collected after 48 hrs. Each point on the graph represents mean ± S.D. of 3 independent readings. (B) MTT assay showing effect of CWR-R1 cells with GCP or vehicle in FBS and CSS containing medium. Each point on the graph represents mean ± S.D. of 3 independent readings. (C) Cell cycle analysis by flow cytometry in PI stained, ethanol-fixed cells demonstrating that CWR-R1 cells treated with 100 μg/ml GCP for 48 hrs undergo G2/M arrest. (D) Immunoblots to determine levels of various FlnA fragments in CWR-R1 cells cultured in FBS vs. CSS and treated with either vehicle or 100 μg/ml GCP for 48 hrs. An antibody to the C-terminal end of FlnA which recognizes the 280, 110 and 90 kDa bands was used in this study. (E) Apoptosis in LNCaP cells determined by flow cytometry in Annexin V and PI-stained in medium containing FBS or CSS in the presence of vehicle or 100 μg/ml GCP for 48 hrs. (F) Role of FlnA in GCP-induced apoptosis. LNCaP cells were subjected to control or FlnA siRNA and treated with vehicle or 100 μg/ml GCP for 48 hrs. Apoptosis was determined by flow cytometry in cells stained with propidium iodide and Annexin V.

Figure 4. A combination of genistein and daidzein are required for rendering the effects of GCP

(A) MTT assay showing the effect of 10 μg/ml genistein or daidezein on CWR-R1 cells grown in either FBS or CSS containing medium. Each point on the graph represents mean ± S.D. of 3 independent readings. (B) Cell cycle analysis by flow cytometry in PI-stained, ethanol-fixed PC-346C cells demonstrating that treatment for 48 hours with GCP and genistein undergo G2/M arrest while cells treated with Daidzein do not. Note that the combination of daidzein (10 μg/ml) and genistein (10 μg/ml) is equivalent in effect to 100 μg/ml GCP, whereas the combination of daidzein (20 μg/ml) and genistein (20 μg/ml) is equivalent in effect to 200 μg/ml GCP. (C) Western blots demonstrating that 48 hour treatment with genistein and GCP, but not daidzein, increases expression of the 90kDa FlnA fragment in C4-2 cells. An antibody to the C-terminal end of FlnA which recognizes the 280, 110 and 90 kDa bands was used in this study. (D) To ensure that the cell lines we used (other than CWR22Rv1 and CWR22R1) do not carry murine retroviruses, we used a Gag antibody (Paprotka et al. 2011) which detect all murine retroviral gag proteins, thus offering a highly sensitive way of detecting viral infection. The cell lysates from the five cell lines were western blotted with Gag and β-actin antibody. As expected, CWR22Rv1 and R1 express murine retroviral gag proteins, whereas others do not.

Figure 5. GCP treatment sensitized castration-sensitive LNCaP cells to androgen withdrawal induced apoptosis by increasing levels of 90 kDa FlnA

(A) MTT assay in LNCaP cells showing that GCP reduced cell numbers in low-androgen media. Cells were plated in medium containing either FBS (high-androgen) or CSS (low-androgen) in the presence of vehicle (50% DMSO, 50% ethanol) or 100 μg/ml GCP for up to 7 days. Each point on the graph represents mean ± S.D. of 3 independent readings. (B) Cell cycle analysis by flow cytometry in propidium iodide (PI) stained ethanol-fixed cells demonstrating that 100 μg/ml GCP induced cell cycle arrest in LNCaP cells cultured for 48 hrs in medium with CSS, but not FBS. In the presence of FBS, GCP induced an increase in cells in G2, only, while in CSS additionally, there was a lack of cells in S-phase. (C, D) Immunoblots to determine levels of various proteins in (C) LNCaP and (D) C4-2 cells cultured in FBS vs CSS in vehicle or 100 μg/ml GCP for 48 hrs. Note that an antibody to the N-terminal end of FlnA was used to determine the 280 kDa band while an antibody to the C-terminal end of FlnA was used to determine the 110 and 90 kDa bands. (E) MTT assay showing growth arrest in C4-2 cells treated with 10 μM bicalutamide and 20 μg/ml GCP despite the continued growth in GCP alone. Each point on the graph represents mean ± S.D. of 3 independent readings.

Figure 6. ADT prevents Filamin A cleavage to the 90 kDa fragment and phosphorylates FlnA at Ser 2152

(A) LNCaP cells were cultured for up to 15 days in FBS or CSS containing medium, and the levels of 90 and 280 kDa FlnA were determined by western blotting. (B) Western blot showing decreased expression of the 90 kDa FlnA fragment in LNCaP cells in CSS vs FBS, whereas its levels were restored by the addition of 1 nM DHT. (C) FlnA expression in CWR22Rv1 cells is regulated by the AR. AR siRNA decreased FlnA mRNA expression by ∼60% as determined by qPCR in medium with CSS (AD-). (D) LNCaP cells were grown in either FBS or CSS containing medium, and FlnA protein expression was determined by immunofluorescence. Scale bar: 10 μm. Note that cells grown in FBS containing medium show higher expression of FlnA compared to cells grown in CSS containing medium. (E) LNCaP and C4-2 cells were cultured in FBS or CSS with increasing levels of DHT as indicated for 48 hrs. Western blots demonstrate the levels of expression of various proteins. Note that C4-2 cells actually have much lower levels of FlnA 90 kDa, therefore the blot shown here for C4-2 was at a higher exposure. (F) (Upper) Scheme showing FlnA cleavage at H1 is regulated by phosphorylation at Ser 2152. When FlnA is phosphorylated, it does not undergo cleavage and remains in the cytoplasm, whereas upon dephosphorylation, FlnA is cleaved to the 90 kDa fragment which translocates to the nucleus. (Lower) LNCaP cells were cultured in FBS or CSS with increasing levels of the PKA inhibitor PKI 14-22 (PKI) for 48 hrs. Western blotting demonstrated that culture in CSS depleted levels of the 90 kDa FlnA, but treatment with increasing doses of PKA inhibitor in CSS containing medium restored its expression.

Figure 7. GCP prevents phosphorylation of full-length FlnA and promotes cleavage of FlnA to its 90 kDa fragment

(A) (Upper) Western blot showing expression of FlnA in CWR22Rv1 cells. Treatment with 10 μM bicalutamide depleted levels of 90 kDa FlnA while transfection with FlnA 16-24 and further treatment with 100 μg/ml GCP for 48 hrs rescued the expression of the 90 kDa FlnA fragment. Note higher exposure of left panel compared to right. (Lower) LNCaP and C4-2 cells treated with vehicle or 100 μg/ml GCP were stained with an antibody specific to the C-terminus FlnA to determine subcellular localization. LNCaP cells and C4-2 cells treated with GCP showed FlnA expression in the nucleus while C4-2 cells treated with vehicle showed expression only in the cytosol. Scale bar: 10 μm. (B) Western blot comparing levels of phosphorylated full-length FlnA (Ser 2152) in various cell lines. (C) Western blot demonstrating that GCP prevented phosphorylation of full-length FlnA and increased expression of 90 kDa FlnA. (D) LNCaP cells, and C4-2 cells transfected with vector or FlnA 16-24 were stained with an antibody specific for phosphorylated FlnA (Ser 2152) and FlnA phosphorylation levels determined by immunofluorescence. Scale bar: 10 μm. (E) LNCaP Cells were cultured in medium containing either FBS or CSS and treated as indicated. Growth in CSS containing medium increased the phosphorylation of full-length FlnA while treatment with GCP reduced full-length FlnA phosphorylation.

Figure 8. GCP treatment prevented relapse in the CWR22 xenograft mouse model

(A) The tumors were paraffin-embedded, sectioned and stained with anti-Ki67 antibody to determine the effect of GCP and castration on proliferation. Note that tumors in sham-operated animals were positive for Ki67 staining whereas those that were castrated, either in the presence or absence of GCP lacked this staining. Tumors were collected 3 days after the operation. (B) FlnA and AR staining (brown) in tumors from animals who were (i) sham operated, untreated (ii) castrated, untreated (iii) castrated, vehicle treated and (iv) castrated, GCP-treated. Note high FlnA staining in the latter group. (C) Both vehicle and GCP-treated tumors rapidly underwent tumor regression within 7 days following castration. Waterfall plot showing tumor regression in the animals 7-days post castration. Note that GCP-treated tumors underwent greater levels of regression compared to those in vehicle-fed mice. Red circle denotes one tumor that did not undergo significant change in volume. (D) After the initial regression (up to ∼35 days) tumor volume remained essentially steady until 179 days post-castration after which GCP-treated tumors continued to regress while vehicle-treated tumors began to recover, and were distinctly larger than the tumors in GCP-treated mice after 211 days.

Figure 8. GCP treatment prevented relapse in the CWR22 xenograft mouse model

(A) The tumors were paraffin-embedded, sectioned and stained with anti-Ki67 antibody to determine the effect of GCP and castration on proliferation. Note that tumors in sham-operated animals were positive for Ki67 staining whereas those that were castrated, either in the presence or absence of GCP lacked this staining. Tumors were collected 3 days after the operation. (B) FlnA and AR staining (brown) in tumors from animals who were (i) sham operated, untreated (ii) castrated, untreated (iii) castrated, vehicle treated and (iv) castrated, GCP-treated. Note high FlnA staining in the latter group. (C) Both vehicle and GCP-treated tumors rapidly underwent tumor regression within 7 days following castration. Waterfall plot showing tumor regression in the animals 7-days post castration. Note that GCP-treated tumors underwent greater levels of regression compared to those in vehicle-fed mice. Red circle denotes one tumor that did not undergo significant change in volume. (D) After the initial regression (up to ∼35 days) tumor volume remained essentially steady until 179 days post-castration after which GCP-treated tumors continued to regress while vehicle-treated tumors began to recover, and were distinctly larger than the tumors in GCP-treated mice after 211 days.