Transcriptional activation by NFκB increases perlecan/HSPG2 expression in the desmoplastic prostate tumor microenvironment

Abstract

Perlecan/HSPG2, a heparan sulfate proteoglycan typically found at tissue borders including those separating epithelia and connective tissue, increases near sites of invasion of primary prostatic tumors as previously shown for other proteins involved in desmoplastic tissue reaction. Studies of prostate cancer cells and stromal cells from both prostate and bone, the major site for prostate cancer metastasis, showed that cancer cells and a subset of stromal cells increased production of perlecan in response to cytokines present in the tumor microenvironment. In silico analysis of the HSPG2 promoter revealed two conserved NFκB binding sites, in addition to the previously reported SMAD3 binding sites. By systematically transfecting cells with a variety of reporter constructs including sequences up to 2.6 kb from the start site of transcription, we identified an active cis element in the distal region of the HSPG2 promoter, and showed that it functions in regulating transcription of HSPG2. Treatment with TNF-α and/or TGFβ1 identified TNF-α as a major cytokine regulator of perlecan production. TNF-α treatment also triggered p65 nuclear translocation and binding to the HSPG2 regulatory region in stromal cells and cancer cells. In addition to stromal induction of perlecan production in the prostate, we identified a matrix-secreting bone marrow stromal cell type that may represent the source for increases in perlecan in the metastatic bone marrow environment. These studies implicate perlecan in cytokine-mediated, innate tissue responses to cancer cell invasion, a process we suggest reflects a modified wound healing tissue response co-opted by prostate cancer cells.

Keywords: CYTOKINES; GENE EXPRESSION; PERLECAN/HSPG2; PROSTATE CANCER; TGFβ; TNF-α.

Figure 1. Perlecan accumulates in the reactive stroma of prostate cancer

Perlecan staining (brown) with nuclear counterstain (blue) in Gleason grade 4 prostate cancer (PCa, right panel) and normal adjacent tissue (Normal, left panel) from the same patient. Increased deposition of perlecan is observed in the regions surrounding PCa when compared to regions of normal morphology. Quantification of perlecan staining in the tissue microarray is shown in figure 1B. This microarray included 34 sections of normal prostate and 23 sections of prostate cancer. Perlecan staining in normal stroma was compared to perlecan staining in tumor stroma in each of these sections. Perlecan staining was significantly higher in tumor stroma than in normal stroma with a p value of 0.0018. Scale bar = 100 µm.

Figure 2. Expression of HSPG2 mRNA and perlecan increase in HS-27a and LNCaP cells during TGFβ1 and TNF-α treatment

Medium was collected and perlecan content assessed by quantitative dot blot as described in Materials and Methods (A). The fold change in perlecan secretion shown in (A) was divided by the live cell number to determine perlecan secretion/cell (B). A time-course treatment was performed using LNCaP cells and cells were collected every 24 hr for 72 hr. Steady state HSPG2 mRNA levels were assessed (C) by qRT-PCR as described in Materials and Methods. Conditioned medium was collected from LNCaP cells over the same time periods, and protein assessed by dot blot as above (D). Data is presented as the ratio of densitometric value of treated medium over the value for vehicle controls set to 1.0 (A,B,D) or Delta delta Ct (C). Bars represent the mean +/− SEM, *, p<.05 vs control, **, p<.01 vs control, ***, p<.001 vs control. At least three biological replicates were performed for each condition.

Figure 3. Conserved NFκB consensus elements are present in the HSPG2 promoter

The schematic depicts the HSPG2 promoter that was used in the luciferase promoter-reporter assays. The full-length reporter used in these studies (A) extends more than 2.6 kb upstream of the transcriptional start site, and includes two putative NFκB binding sites in the most distal 200 base pairs. The 2.4 kb deletion construct (B) was created by removing the distal 200 base pairs of the 2.6 kb promoter. Directed mutants of the putative NFκB sites include mutation of NFκB 1 (C), NFκB 2 (D) or both NFκB sites (E).

Figure 4. HSPG2 promoter activity increases in response to TNF-α in a dose-dependent manner

Cells were transfected with the 2.6 kb reporter plasmid shown in 3A, and treated for 24 hr with TNF-α at the indicated final concentrations (A), before measuring luciferase activity in lysed cells as described in the text. Concentrations above10 ng/ml did not further increase activity (not shown). For experiments shown in (B), cells were treated with TGFβ1 (10 ng/ml), TNF-α (10 ng/ml) or a combination of the two, then luciferase activity was measured as described in Materials and Methods. Data are reported as the fold change observed between cytokine treated and vehicle treated cells. Bars represent the mean +/− SEM, *, p<.05 vs .01, control, **, p<.001 vs .1, .01, control, ***, p<.001 vs 1, 0.1, 0.01, control. At least three biological replicates were performed for each condition.

Figure 5. TNF-α treatment triggers nuclear translocation of NFκB in HS-27a and WPMY-1, but not HS-5, cells

Cells were treated with TNF-α (10 ng/mL) in serum-free medium for 1 hr, permeabilized, and then stained with anti-NFκB as described in Materials and Methods. Cells were imaged by confocal microscopy. Red, NFκB. Green, phalloidin. Blue, DAPI. Scale bars, 50 µm.

Figure 6. Deletion of the distal 200 base pairs of the HSPG2 promoter or mutation of NFκB consensus elements eliminates responsiveness to TNF-α and TGFβ1

Cells in culture were transfected with the 2.4 kb promoter seen in Figure 3B, or constructs in which the two NFκB sites were mutated as described above and responses to each cytokines alone (10 ng/ml) or in combination (each at 10 ng/ml) were measured at 24 hr of treatment. Panel A shows that removal of the entire region containing both sites eliminates responses to cytokines in all four cell lines shown. Panel B shows that 2.6_mut1 or 2.6_mut1,2, but not 2.6_mut2, loses activity, hence response to TNF-α is associated with the more distal of the two putative NFκB sites. Bars represent the mean +/− SEM. **, p<.01 vs wild-type, ***, p<.001 vs wild type. At least three biological replicates were performed for each condition.

Figure 7. p65 binds the HSPG2 5' regulatory region in LNCaP cells during TNF-α treatment

Chromatin immunoprecipitation was used to detect p65 binding the perlecan promoter. The HSPG2 regulatory region is significantly enriched compared to the background control (the HBB promoter) in LNCaP cells treated with TNF-α. Endpoint PCR (panel A) indicates the HSPG2 regulatory region is amplified when p65 is pulled down in the chromatin of TNF-α treated cells, but not in that of cells treated with the vehicle control. This amplification was quantified using quantitative PCR. The HSPG2 regulatory region was also pulled down in Ishikawa and Hec-50 endometrial cancer cells, used as positive controls for NFκB activation. Three biological replicates were performed using LNCaP cells, and one replicate was used for each of the endometrial cancer cell lines.

Figure 8. Model for HSPG2 induction by tumor and immune cell interaction

In our proposed model, tumor recruitment of immune cells including macrophages results in inflammatory cytokine release. This amongst other cancer cell-derived factors induces stromal activation. Part of this phenotypic program is perlecan secretion.