Lumican expression, localization and antitumor activity in prostate cancer

Abstract

The stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. The role of lumican in cancer varies according to the type of tumor. In this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. The increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. In vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin β1 and MT1-MMP, and invadopodia detected by disruption of α-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. In conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging.

Figure 1

Expression and localization of Lumican in human neoplastic and non-neoplastic prostate. Fresh neoplastic and contralateral non-neoplastic specimens were isolated from human prostate primary tumors; the specimens were frozen and stained using hematoxylin/eosin for histological determination of neoplastic and non-neoplastic regions. Following confirmation, neoplastic and non-neoplastic tissues were cut in half; one fragment was allocated for RNA extraction and real-time PCR analysis (A), and the other fragment was fixed with 4% paraformaldehyde for immunohistochemistry assays (B and C). An increase in lumican expression was observed both through real-time PCR (A) and immunohistochemistry (B and C). Lumican in neoplastic tissues displayed characteristic fibrotic disposition surrounding neoplastic acinar glands. Gene expression was normalized against ribosomal protein S29 (RPS29). *p<0.05.

Figure 2

Effect of Lumican on prostate cancer cell proliferation. Prostate cancer cells (PC3 (A), DU145 (B) and LnCAP (C)) were treated with increasing concentrations of lumican extracted from amniotic membrane for 24 h. Cell proliferation was measured using the BrdU assay. Treatment of cells with anti-lumican blocked the effect of lumican on cell proliferation. Error bars indicate S.D. of six samples. (*) p<0.05 versus control (0 µg/mL).

Figure 3

Role of lumican on prostate cancer cell migration. 24 well polystyrene culture dishes were incubated in a solution of 10 µg/mL lumican and left for 2 h in a sterile atmosphere (flow hood) and then washed 3 times in EBSS. Untreated culture dishes were used as controls. After culture dish treatment, 2 × 10⁴ prostate cancer cells (PC3 (A and D), DU145 (B and E) and LNCaP (C and F)) were seeded in RPMI containing L-glutamine/penicillin/streptomycin and 10% FBS, and incubated for 24 h at 37°C in a 5% CO₂ humidified environment. A scratch was performed in the cells on the culture dish using a 100 µL pipette tip. Cells were washed three times with EBSS and maintained for a further 24 h (37°C, 5% CO₂), images being captured every 4 h. Photos were taken using phase contrast. Measurements were calculated using Image-pro.

Figure 4

Role of lumican on prostate cancer cell invasion. The effect of lumican on the invasion of prostate cancer cells was analyzed using culture plate inserts (8 µm pore size) previously treated with 1 µg/mL, 5 µg/mL or 10 µg/mL of lumican purified from amniotic membrane for 2 h and subsequently washed 3 times in EBSS. Control transwell inserts were treated with BSA. After insert treatment, 2 × 10⁴ prostate cancer cells (PC3 (A) and DU145 (B)) were seeded in the insert in RPMI containing L-glutamine/penicillin/streptomycin in the absence of FBS, and incubated for 4 h at 37°C in a 5% CO₂ humidified environment. Thereafter, RPMI enriched with 10% FBS was added to the bottom compartment and the cells were incubated for a further 2 h (37°C, 5% CO₂), and subsequently the inserts were washed, fixed and stained with crystal violet solution (0.2% in distilled water). Cells in the top compartment were removed using a Q-tip, and migrated cells (C) were counted (A and B).

Figure 5

Role of lumican on keratin 8 and 18 filaments in prostate cancer cells. Coverslips placed in 24 well polystyrene culture dishes were incubated in a solution of lumican (10 µg/mL) and untreated culture dishes were used as controls. After culture dish treatment, 2 × 10⁴ prostate cancer cells, PC3, were seeded in RPMI containing L-glutamine/penicillin/streptomycin and 10% FBS, and incubated for 24 h at 37°C in a 5% CO₂ humidified environment. A scratch was performed in the cells on the culture dish using a 100 µL pipette tip. Cells were washed three times with EBSS, maintained for a further 4 h (37°C, 5% CO₂) and fixed for immunocytochemistry. The cells were labeled with anti-keratin 8 and anti-keratin 18 and the nuclei were visualized with DAPI. Dashed line represents the original wound edge. Images were captured using a Zeiss LSM510 scanning Confocal inverted microscope and Zeiss Observer Z1 microscope coupled with an ApoTome.

Figure 6

Role of lumican on the expression and localization of actin filaments, ZO-1, integrin β1 and MT1-MMP in prostate cancer cells. Coverslips placed in 24 well polystyrene culture dishes were incubated in a solution of lumican (10 µg/mL) and untreated culture dishes were used as controls. After culture dish treatment, 2 × 10⁴ prostate cancer cells, PC3, were seeded in RPMI containing L-glutamine/penicillin/streptomycin and 10% FBS, and incubated for 24 h at 37°C in a 5% CO₂ humidified environment. A scratch was performed in the cells on the culture dish using a 100 µL pipette tip. Dashed line represents the original wound edge. Cells were washed three times with EBSS, maintained for a further 4 h (37°C, 5% CO₂) and fixed for immunocytochemistry. The cells were labeled with phalloidin (A) and anti-ZO-1 (B) and the nuclei were visualized with DAPI. Images were captured using a Zeiss LSM510 scanning Confocal inverted microscope and Zeiss Observer Z1 microscope coupled with an ApoTome.

Figure 7

Role of lumican on the expression and distribution of N-WASP, cortactin and sm α-actin in prostate cancer cells. Coverslips placed in 24 well polystyrene culture dishes were incubated in a solution of either 1 µg/mL collagen I or sequential 1 µg/mL collagen I and 10 µg/mL lumican. After culture dish treatment, 2 × 10⁴ prostate cancer cells, PC3 (A) or DU145 (B), were seeded in RPMI containing L-glutamine/penicillin/streptomycin and 10% FBS, and incubated for 24 h at 37°C in a 5% CO₂ humidified environment. A scratch was performed in the cells on the culture dish using a 100 µL pipette tip. Cells were washed three times with EBSS, maintained for a further 4 h (37°C, 5% CO₂) and fixed for immunocytochemistry. The cells were labeled with anti-N-WASP, anti-sm α-actin, anti-cortactin and DAPI and analyzed using a Zeiss LSM510 scanning Confocal inverted microscope.

Figure 8

Role of lumican on the capacity of prostate cancer cells to invade adjacent tissue. Prostate cancer cells (PC3) previously labeled with DiI (red) were seeded upon peritoneal tissue isolated from either lumican KO mice or littermate heterozygous controls and left to invade for 24 h. The peritoneum was washed and fixed and the invaded cells were visualized under a fluorescent microscope. The total number of DiI positive cells which had penetrated either lumican KO or littermate control tissue were counted and represented in a graph (A). PC3 cells adhered to and invaded the lumican KO peritoneal tissue, whereas few cells adhered to the heterozygous tissue and no cells fully invaded the heterozygous tissue (B). PC3 cellular projections (asterisk) protrude into the lumican KO peritoneal tissue.