Matrilysin/MMP-7 Cleavage of Perlecan/HSPG2 Complexed with Semaphorin 3A Supports FAK-Mediated Stromal Invasion by Prostate Cancer Cells

Abstract

Interrupting the interplay between cancer cells and extracellular matrix (ECM) is a strategy to halt tumor progression and stromal invasion. Perlecan/heparan sulfate proteoglycan 2 (HSPG2) is an extracellular proteoglycan that orchestrates tumor angiogenesis, proliferation, differentiation and invasion. Metastatic prostate cancer (PCa) cells degrade perlecan-rich tissue borders to reach bone, including the basement membrane, vasculature, reactive stromal matrix and bone marrow. Domain IV-3, perlecan's last 7 immunoglobulin repeats, mimics native proteoglycan by promoting tumoroid formation. This is reversed by matrilysin/matrix metalloproteinase-7 (MMP-7) cleavage to favor cell dispersion and tumoroid dyscohesion. Both perlecan and Domain IV-3 induced a strong focal adhesion kinase (FAK) dephosphorylation/deactivation. MMP-7 cleavage of perlecan reversed this, with FAK in dispersed tumoroids becoming phosphorylated/activated with metastatic phenotype. We demonstrated Domain IV-3 interacts with the axon guidance protein semaphorin 3A (Sema3A) on PCa cells to deactivate pro-metastatic FAK. Sema3A antibody mimicked the Domain IV-3 clustering activity. Direct binding experiments showed Domain IV-3 binds Sema3A. Knockdown of Sema3A prevented Domain IV-3-induced tumoroid formation and Sema3A was sensitive to MMP-7 proteolysis. The perlecan-Sema3A complex abrogates FAK activity and stabilizes PCa cell interactions. MMP-7 expressing cells destroy the complex to initiate metastasis, destroy perlecan-rich borders, and favor invasion and progression to lethal bone disease.

Figure 1

Full length perlecan induces clustering in prostate cancer cells over time. C4-2B cells were seeded into wells coated with perlecan (A–C), BSA (D–F), or tissue culture plate only (G–I) and imaged over a 96-hour period. Cells on perlecan begin clustering at one hour and is more pronounced later (15 and 96 hours) (white boxed inset enlarges clusters). Cells seeded on BSA coated plates lack any observable difference between the plate only, becoming adherent and spreading out in both cases. Scale bars: 100 µm.

Figure 2

Heparanase/chondroitinase and MMP-7 enzymatic processing of perlecan favors cell substratum versus cell-cell adhesion. C4-2B cells were seeded onto various types of perlecan and control samples pre-adsorbed to the plate and imaged by bright field microscopy and then stained for F-actin (green) and nuclei (blue) after 1 hour. Perlecan digested with a mixture of heparitinase and chondroitinase ABC (H/C) (panels D–F) or MMP-7 (panels G–I) adhered to the plates within one hour and remained attached through the staining procedure (E,F and H,I). Neither intact perlecan (A), MMP-7 alone (B), nor H/C mixture alone (C) supported this rapid adherence to substratum, and cells remained round or formed loose clusters. Boxed insets provide an enlarged image to show cell morphology. Scalebars: 100 µm for A-D and G; 50 µm for E and H; 25 µm for F and I.

Figure 3

Perlecan Domain IV-3 mediates a strong repulsive/clustering effect on C4-2 cells as does full length perlecan. (A) Schematic of full length perlecan highlighting Domain IV-3 (the last 7 Ig repeats of Domain IV). A reverse phase protein array (RPPA) was performed on cells cultured on wells entirely coated with Domain IV-3 (panel B) or MMP-7 cleaved Domain IV-3 (panel C) for 48 hours. Cells cluster on Domain IV-3 and this is reversed by pre-cleavage with MMP-7. D.) C4-2 cells 72 hours after being cultured on a spot (inside the white dashed circle) of surface adsorbed Domain IV-3. Cells cleared out of the area, creating a border around Domain IV-3. Scalebars are 250 µm. White asterisks indicate fiduciary markers (black areas) used to approximate the location of the spotted Domain IV-3 at the time.

Figure 4

Full length perlecan and Domain IV-3 strongly dephosphorylate focal adhesion kinase (FAK) and alter several downstream signaling components. (A) C4-2 cells were cultured on several substrates (Set 1: Control BSA/digest buffer, set 2: Domain IV-3 alone, set 3: Domain IV-3 + MMP-7, set 4: perlecan + MMP-7, and set 5: perlecan alone) for 24 hours and probed for phosphorylated FAK (p-FAK), total FAK, p-AKT, FoxM1, p-p38, and GAPDH. Unpaired student t-tests between sets 2 and 3 were performed for densitometry quantified values of p-FAK/FAK, p-AKT/AKT, FoxM1/GAPDH, and p-p38/GAPDH. (B) A 12, 24 and 48-hour time course of C4-2 cells cultured on Domain IV-3 incubated either alone (−) or with MMP-7 (+) and probed for signaling components that were quantified by densitometry. An unpaired student’s t-test measured any significant difference between cells on Domain IV-3 or Domain IV-3 pre-cleaved with MMP-7 at each time point. p-values: * < 0.05, ** < 0.01, *** < 0.001. Expanded view of blots included in composite figure included in supplemental data file.

Figure 5

Semaphorin 3A (Sema3A) antibody imitates Domain IV-3 activity on prostate cancer cells. Cell culture wells were spotted with control BSA and rabbit IgG (panel A), Domain IV-3 and rabbit IgG (panel B), BSA and Sema3A antibody (Ab) (panel C), or Domain IV-3 and Sema3A antibody (panel D) (inside the white dashed arc line). C4-2 cells were seeded and imaged 24 hours later. Scalebar is 250 µm. White asterisks indicate black fiduciary markers used to approximate the area of surface adsorbed substrate. (B) A western blot of C4-2 cells cultured for 24 hours on Domain IV-3 (lane 1), rabbit IgG (lane 2), Sema3A antibody (lane 3), or control BSA (lane 4). (C) Western blot of C4-2 cells cultured for 24 hours on control rabbit IgG or Sema3A antibody and probed for p-FAK, FAK, p-ERK, ERK1/2, and GAPDH. The Sema3A antibody as a substrate produces results akin to Domain IV-3 for C4-2 cells.

Figure 6

Sema3A binds Domain IV-3 and shRNA against Sema3A abrogates C4-2 cell clustering. (A) Domain IV-3 was adsorbed to well surfaces and subjected to a binding assay with Sema3A-Fc, Sema3A-Fc and Sema3A antibody, Sema3A antibody alone, and control IgG. Sema3A-Fc binds to Domain IV-3 and Sema3A antibody diminishes the binding signal significantly to 250 ng/mL (student’s unpaired t-test per concentration). (B) Blossom62 alignment of Ig 17 of perlecan (2^nd of Domain IV-3) and the Ig of Sema3A. C4-2 cells were seeded on Domain IV-3 after being transduced with either control shRNA (panel C) or Sema3A directed shRNA (panel D). (E) Dispersion index quantification between control and directed shRNA for each substrate type and amount. Significance values are student’s unpaired t-test between control and Sema3A shRNA. p-values: *<0.05, **<0.01, ***<0.001.

Figure 7

Recombinant Sema3A-Fc is digested by MMP-7. (A) MMP-7 in silico digestion of Sema3A using SitePrediction online software. Shown is a schematic of Sema3A with its domains (sema, plexin/semaphorin/integrin (PSI), immunoglobulin (Ig)-like, basic). Each line represents a cleavage site with > 95% specificity. The bolded number is the rank in average score. Below rank average score is the amino acid sequence with the P1 cleavage site (cleaves at period). Amino acid sequence is based on UniProt ID Q14563, which includes the signal sequence not shown in the schematic. (B) Sema3A-Fc (0.75 µg) was incubated alone or with MMP-7 (0.08 µg) overnight. A silver stain and western blot against Sema3A indicate MMP-7 fragments Sema3A into multiple smaller sub bands including a 30 kDa band indicated by an arrow.

Figure 8

Model of perlecan/Sema3A proposed interactions. In non-invasive prostate overgrowth or early cancer (PCa), intact perlecan is present in the immediate stroma surrounding Sema3A expressing prostatic cells. In this state (left), perlecan engages Sema3A, stabilizing its interaction with the neuropilin-1 and plexin complex. Plexin Ras-GAP activity favors a state of focal adhesion kinase (FAK) dephosphorylation and integrin deactivation. In this state, PCa cell-cell adhesion dominates cell-substratum adhesion, abrogating tissue invasion. As PCa progresses, active MMPs such as MMP-7 can cleave perlecan and/or Sema3A. Ras-GAP activity of plexins is lost, allowing FAK phosphorylation to occur along with subsequent downstream signaling (right). FAK signaling reactivates integrins to favor substrata-mediated dispersion, as seen to occur during tumor dyscohesion and metastasis. GAP = guanosine triphosphate (GTP)ase activation protein.