A holistic approach to dissecting SPARC family protein complexity reveals FSTL-1 as an inhibitor of pancreatic cancer cell growth

Abstract

SPARC is a matricellular protein that is involved in both pancreatic cancer and diabetes. It belongs to a wider family of proteins that share structural and functional similarities. Relatively little is known about this extended family, but evidence of regulatory interactions suggests the importance of a holistic approach to their study. We show that Hevin, SPOCKs, and SMOCs are strongly expressed within islets, ducts, and blood vessels, suggesting important roles for these proteins in the normal pancreas, while FSTL-1 expression is localised to the stromal compartment reminiscent of SPARC. In direct contrast to SPARC, however, FSTL-1 expression is reduced in pancreatic cancer. Consistent with this, FSTL-1 inhibited pancreatic cancer cell proliferation. The complexity of SPARC family proteins is further revealed by the detection of multiple cell-type specific isoforms that arise due to a combination of post-translational modification and alternative splicing. Identification of splice variants lacking a signal peptide suggests the existence of novel intracellular isoforms. This study underlines the importance of addressing the complexity of the SPARC family and provides a new framework to explain their controversial and contradictory effects. We also demonstrate for the first time that FSTL-1 suppresses pancreatic cancer cell growth.

Figure 1. Domain structure of the SPARC family of proteins.

The SPARC family of proteins share three main domains: domain I- a highly acidic region with low affinity calcium binding, domain II- a follistatin-like domain containing kazal-like serine protease inhibitor domains, and domain III- a calcium binding EF hand domain (also referred to as the EC domain). The signal peptide (indicated by red boxes) is typically located in domain I except for SMOC proteins. Individual members of the SPARC family also have distinct domains: SPOCK proteins contain a thyroglobulin domain and a glycosaminoglycan binding domain, while SMOC proteins contain 2 thyroglobulin domains. FSTL-1 contains a von Willebrand factor type-C domain.

Figure 2. The SPARC family of proteins are widely expressed in the pancreas.

ICR mouse pancreas sections were probed with antibodies to: Hevin (a), SPOCK-1 (b), and SPOCK-2 (c), followed by ABC-DAB staining (brown) and counterstained with haematoxylin (blue). Images are representative of 3–5 islets and ducts per section acquired using a 20X objective. Scale bar 100 μm. n = 2–3 independent experiments from 3 different mouse pancreas. See main text for explanation of arrows.

Figure 3. The SPARC family of proteins are widely expressed in the pancreas.

ICR mouse pancreas sections were probed with antibodies to: SPOCK-3 (a), SMOC-1 (b), SMOC-2 (c). See Figure legend 2 for additional details.

Figure 4. The SPARC family of proteins are widely expressed in the pancreas.

ICR mouse pancreas sections were probed with antibodies to FSTL-1. See Figure legend 2 for additional details.

Figure 5. Identification of multiple isoforms of the SPARC family and their expression in specific cell types.

For each of the indicated cell types 20–25 μg of protein lysate was analysed by western blot using antibodies to Hevin (a), SPOCK-1 (b), SPOCK-2 (c), SPOCK-3 (d), SMOC-1 (e), FSTL-1 (f). Blots were cropped to show consistent bands observed in at least 2 independent experiments. Full, uncropped blots are shown in Suppl. Figure 2.

Figure 6. Predicted domain structures of alternative splice variants of the extended SPARC family.

FASTA format protein sequences of the SPARC family alternative transcripts (complete CDS) were obtained from ENSEMBL and the InterPro database was used to determine domain structures. Numbers within domain structures indicate amino acid residues for each domain. Red boxes indicate the presence of a signal peptide. Alternative transcripts that do not contain the signal peptide are indicated with asterisks.

Figure 7. Hevin contains N-linked glycosylated isoforms, but expression is not regulated by loss of SPARC expression.

(a) PS-1 cell lysates were treated with PNGase-F glycosidase and analysed by western blot using antibodies to Hevin and β actin as a loading control. Images are representative of 3 independent replicates, and the mean molecular weight observed for each band is indicated next to the blots. Arrow indicates de-glycosylated band. (b–e) PS-1 stellate cells were treated with anti-SPARC (+) or control (−) siRNA for 48 hours. Cell lysates were then analysed by western blot using antibodies to SPARC to confirm knockdown (b), Hevin N-terminus (c), or Hevin C-terminus (d). On average 90% knockdown of SPARC expression was achieved in the three experiments performed (+/− 6%). In (e), the graph shows mean signal intensity +/− SEM for the Hevin N-terminal antibody, standardised to β actin and relative to the control, for cells treated with SPARC (KD) or control siRNA. Images are representative of 3 independent replicates, and statistical significance was measured using the Student’s t-test (unpaired, two-tailed). p-values are indicated in the graph. Uncropped blots are shown in Suppl. Figure 4.

Figure 8. Expression of SPOCK-3 splice variants in pancreatic stellate cells.

(a) PS-1 cell lysates were treated with PNGase-F glycosidase and analysed by western blot using antibodies to SPOCK-3 and β actin as a loading control. Images are representative of 3 independent replicates, and the mean molecular weight observed for each band is indicated next to the blots. Arrows indicate de-glycosylated bands. Uncropped blots are shown in Suppl. Figure 5. (b) PS-1 mRNA was isolated and expression of the indicated SPOCK-3 splice variant expression was analysed by RT-PCR.

Figure 9. FSTL-1 does not regulate β cell growth and proliferation.

INS-1 β-cells were plated at a density of 1.5 × 10⁴ cells/well in a 96 well plate. Post synchronisation, the cells were either provided with untreated medium or medium supplemented with 100 ng/ml rFSTL-1 in either 10% FBS (a,b) or 0.5% FBS (c,d) and cultured for a further 72 hrs. Cell growth was monitored every 12 hrs during this period using the IncuCyteZOOM live cell imaging system. In (a,c) graphs shows mean cell confluence relative to control +/− SEM, n = 30 from 5 independent experiments, while representative images taken at the indicated timepoints are shown in (b,d). BrdU incorporation was measured for the last 24 hrs of the 72 hr culture, and the graph in (e) shows mean absorbance relative to the control +/− SEM. Statistical significance was measured using Student’s t-test (unpaired, two-tailed) and p-values are indicated in the graph.

Figure 10. FSTL-1 inhibits pancreatic cancer cell growth and proliferation.

AsPC-1 cancer cells were plated at a density of 5 × 10³ cells/well in a 96 well plate. Post synchronisation, the cells were cultured in complete media (10% FBS) containing the indicated concentrations of rFSTL-1 for 72 hrs. Cell growth was monitored every 12 hrs during this period using the IncuCyteZoom live cell imaging system. (a) Graph shows mean cell confluence relative to the control +/− SEM, n = 17–18 data pooled from 3 independent experiments, and representative images taken at 72 hrs are shown in (b). BrdU incorporation was measured for the last 24 hrs and the graph in (c) shows the mean absorbance relative to the control +/− SEM n = 16–17 data pooled from 3 independent experiments. Statistical significance was measured using one-way ANOVA and p-values are indicated in the graph (*p < 0.05, ^◊p < 0.01, ^†p < 0.001, ^‡p < 0.0001). (d) FSTL-1 expression in pancreatic cancer cells was analysed by western blotting. Uncropped blots are shown in Suppl. Figure 6. (e) RT-PCR was performed on the indicated cell lines using primers specific for DIP2A, or QARS as a housekeeping gene. A single PCR product of the expected size was observed for both primer pairs, and all negative controls were blank.