Expression of the preadipocyte marker ZFP423 is dysregulated between well-differentiated and dedifferentiated liposarcoma

Abstract

Background: Well-differentiated and dedifferentiated liposarcomas are rare soft tissue tumors originating in adipose tissue that share genetic abnormalities but have significantly different metastatic potential. Dedifferentiated liposarcoma (DDLPS) is highly aggressive and has an overall 5-year survival rate of 30% as compared to 90% for well-differentiated liposarcoma (WDLPS). This discrepancy may be connected to their potential to form adipocytes, where WDLPS is adipogenic but DDLPS is adipogenic-impaired. Normal adipogenesis requires Zinc Finger Protein 423 (ZFP423), a transcriptional coregulator of Perixosome Proliferator Activated Receptor gamma (PPARG2) mRNA expression that defines committed preadipocytes. Expression of ZFP423 in preadipocytes is promoted by Seven-In-Absentia Homolog 2 (SIAH2)-mediated degradation of Zinc Finger Protein 521 (ZFP521). This study investigated the potential role of ZFP423, SIAH2 and ZFP521 in the adipogenic potential of WDLPS and DDLPS.

Methods: Human WDLPS and DDLPS fresh and paraffin-embedded tissues were used to assess the gene and protein expression of proadipogenic regulators. In parallel, normal adipose tissue stromal cells along with WDLPS and DDLPS cell lines were cultured, genetically modified, and induced to undergo adipogenesis in vitro.

Results: Impaired adipogenic potential in DDLPS was associated with reduced ZFP423 protein levels in parallel with reduced PPARG2 expression, potentially involving regulation of ZFP521. SIAH2 protein levels did not define a clear distinction related to adipogenesis in these liposarcomas. However, in primary tumor specimens, SIAH2 mRNA was consistently upregulated in DDLPS compared to WDLPS when assayed by fluorescence in situ hybridization or real-time PCR.

Conclusions: These data provide novel insights into ZFP423 expression in adipogenic regulation between WDLPS and DDLPS adipocytic tumor development. The data also introduces SIAH2 mRNA levels as a possible molecular marker to distinguish between WDLPS and DDLPS.

Keywords: Adipogenesis; Liposarcoma; PPARgamma; SIAH2; ZFP423; ZFP521.

Fig. 1

Adipogenesis in LPS tissues is dysregulated at preadipocyte commitment. A Demographic information available for tissues included in the analysis. B Western blots analysis of SIAH2, ZFP423, PPARG, and β-actin. Biological replicates of western blot analyses are available in Supplemental Fig. S1, along with full-length original western blots and densitometry ratio quantifications. C Gene expression of markers of mature adipocytes (PPARG2, ADIPOQ, PLIN1), preadipocyte markers (PREF-1, PDGFRA, ZFP423) or regulatory factors involved in adipogenesis (CEBPB, CEBPD, ZFP521, SIAH2). D Gene expression of macrophage (CD11B, CD64) and cytokine (IL6, IL10) markers. Each point in bar plot represents a single technical replicate from frozen normal human retroperitoneal adipose tissues (rpWAT, n = 3), well-differentiated liposarcoma (WD, n = 2), and dedifferentiated (DD, n = 2). Bar plot represents mean ± standard deviation of fold change when compared to rpWAT; *, p < 0.05; **, p < 0.001; ***, p < 0.0001. N/A, not available; UD, undetermined; AA/Black, African American/Black; W, White

Fig. 2

SIAH2 mRNA is upregulated in DDLPS compared to WDLPS and rpWAT. A Hematoxylin and eosin (H&E) stained DDLPS, WDLPS, and rpWAT. B In situ hybridization detection of SIAH2 (yellow) and immunohistochemistry detection of nuclei (DAPI, blue) and adipocytes using PLIN1 (red) in DDLPS, WDLPS, and rpWAT. Section of the image, marked by the white square, was enlarged to aide with SIAH2 visualization. The images shown are representative of N = 3 for rpWAT, N = 4 for WDLPS and N = 5 for DDLPS

Fig. 3

DDLPS is more fibrotic and heterogeneous with more macrophages compared to rpWAT or WDLPS tissues. A Hematoxylin and eosin (H&E) or trichrome stained DDLPS, WDLPS, and rpWAT. Trichrome stains connective tissue, an indication of fibrosis. B In situ hybridization detection of SIAH2 (yellow) and immunohistochemistry detection of nuclei (DAPI, blue) and macrophages using IBA1 (magenta) in DDLPS, WDLPS, and rpWAT. Section of the image, marked by the white square, was enlarged to aide with SIAH2 visualization. The images shown are representative of N = 3 for rpWAT, N = 4 for WDLPS and N = 5 for DDLPS. C The ratio of SIAH2 mRNA to nuclei in the images shown in Figs. 1 and 2B was determined using the ImageJ Analyze Particle function in the corresponding imaging channels for SIAH2 mRNA signals or DAPI staining of nuclei

Fig. 4

ZFP423 mRNA levels correlate with PPARG2 mRNA levels in normal white adipose tissue primary cells or WDLPS and DDLPS cell lines. Gene expression of biomarkers of WDLPS and DDLPS (MDM2, CDK4) and factors involved in adipogenesis of human retroperitoneal adipose primary cell (HuASC), WDLPS (WD), and DDLPS (224, 863, and 815) at pre-induction, day 3 of induction, and day 14 of induction for adipogenesis. Bar plots depict mean ± standard deviation compared to pre-induced HuASC; *, p < 0.05, **, p < 0.01, ***, p < 0.001. Each point in the plot represents a technical replicate. 224, Lipo224; 863, Lipo863; 815, Lipo815. The plots are representative of experiments that were repeated at least twice

Fig. 5

ZFP423 protein expression is associated with upregulation of PPARG2 protein in normal adipose tissue primary cells (HuASC) and DDLPS cell lines. A Oil Red O staining of human retroperitoneal adipose primary cell (HuASC), WDLPS (WD), and DDLPS (224, 863, and 815) after 14 days of adipogenic induction. B Quantification of Oil Red O staining. C Western blot analysis of ZFP521, SIAH2, PPARG, and β-actin from HuASC, WDLPS, and DDLPS cell lines at pre-induction (Day 0), and 3 (Day 3) or 14 days (Day 14) post induction. 224, Lipo224 cell line; 863, Lipo863 cell line; 815, Lipo815 cell line. A full-length, original and unprocessed version of the western blot for each antibody and densitometry ratio quantification is shown in Supplemental Fig. S2. The plots are representative of experiments that were repeated at least twice. Bar plots depict mean ± standard deviation compared to HuASC; *, P < 0.05, ** P < 0.1 and *** P < 0.001

Fig. 6

ZFP423 knockdown attenuates adipocyte differentiation in human ASC and DDLPS Lipo863 cells. A Oil Red O staining of human retroperitoneal adipose primary cells (HuASC) and DDLPS (Lipo863) after 4 days of adipogenic induction with accompanying quantification of Oil Red O staining. B Gene expression of factors involved in adipogenesis at pre-induction (D0) and day 4 (D4) post-induction. C Western blot analysis of ZFP521, SIAH2, PPARG, ZFP423, and β-actin at pre-induction (0) and day 4 [4] post induction. Bar plots depict mean ± standard deviation compared to pre-induced HuASC; *, p < 0.05, **, p < 0.01, ***, p < 0.001. Each point in the plots (A and B) represents a technical replicate. Full-length western blot replicates and densitometry ratio quantifications are shown in Supplemental Fig. S3. Note that the western blots for PPARG1 and PPARG2 detection in S3 (native MW around 50 kD) include very high molecular weight bands that may represent ubiquitin-modified PPARG

Fig. 7

ZFP423 overexpression (OE) promotes adipocyte differentiation in DDLPS Lipo224. A Oil Red O staining of well-differentiated liposarcoma (WDLPS) and DDLPS (Lipo224) after 10 days of adipogenic induction with accompanying quantification of Oil Red O staining. B Gene expression of factors involved in adipogenesis at pre-induction (D0) and day 10 (D10). C Western blot analysis of ZFP521, SIAH2, PPARG, ZFP423, and β-actin at pre-induction (0) and day 10 [10] post-induction. Bar plots depict mean ± standard deviation compared to pre-induced HuASC; *, p < 0.05, **, p < 0.01, ***, p < 0.001. Each point in the plots represents a technical replicate. 224, Lipo224. Full-length western blot replicates and densitometry ratio quantifications are shown in Supplemental Fig. S4. Note that the western blots for PPARG1 and PPARG2 detection in S4 (native MW around 50 kD) include very high molecular weight bands that may represent ubiquitin-modified PPARG