Cell Growth Regulated by Syndecan-1 in Human Liposarcoma

Background: The heparan sulfate proteoglycan, syndecan-1 (also known as CD138), is associated with cell proliferation, adhesion, and migration in various types of human malignancies. The role of syndecan-1 in soft tissue tumors is unknown; therefore we examined whether syndecan-1 plays a role in human liposarcoma. Materials and Methods: We performed immunohistochemical analyses of syndecan-1 in 42 liposarcoma cases, including 21 myxoid liposarcoma cases. Recurrence-free survival was estimated by the Kaplan–Meire method. We examined the association between apoptosis and syndecan-1 gene silencing in a human liposarcoma cell line. Results: The proportion of syndecan-1 positive cells (≥10%) was significantly associated with unfavorable recurrence-free survival in myxoid liposarcoma patients. There were no significant associations between syndecan-1 expression and prognosis in the other subtypes of liposarcoma. Knockdown of syndecan-1 induced apoptosis and suppression of cell growth in vitro. Conclusion: Syndecan-1 could be a novel prognostic factor for myxoid liposarcoma recurrence and a new therapeutic target for human myxoid liposarcoma.

Surgery serves as the sole therapy for localized liposarcomas.Generally, liposarcomas are known to be a relatively chemoresistant tumor, with the exception of the myxoid variant.Conventional chemotherapy with doxorubicin and ifosfamide is the first-line treatment for liposarcomas.Other active cytotoxic agents include gemcitabine, docetaxel and the marine-derived compound trabectedin (Kollár & Benson, 2014).Moreover, liposarcomas, excluding the myxoid variant, are highly resistant to radiotherapy.Liposarcoma patients with metastases or poor response to chemotherapy have worse prognosis; therefore a new therapeutic drug is required.
We conducted a clinicopathologic and prognostic analysis of syndecan-1 expression in 42 liposarcoma specimens and their benign counterparts.We examined the roles of syndecan-1 in liposarcoma by in vitro studies.Syndecan-1 overexpression was correlated to a poor prognostic factor for recurrence-free survival in myxoid liposarcoma patients.In addition, syndecan-1 silencing through siRNA transfection effectively induced cellular apoptosis and growth suppression of liposarcoma cells in vitro.We found that cell growth and clinical recurrence might be enhanced by syndecan-1 in human myxoid liposarcoma.

Cell Culture
The human liposarcoma cell line SW872 was purchased from the American Type Culture Collection (Rockville, MD, USA).Cells were maintained in Leibovitz's L-15 medium with 12% fetal bovine serum at 37 °C/5% CO 2 .

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick end Labeling (TUNEL) Assays
DNA cleavage, a characteristic of apoptosis, was detected using the TUNEL assay (Apop Tag Plus Peroxidase In situ Apoptosis Detection Kit; Millipore, Temecula, CA, USA).After siRNA transfection, cells were washed with phosphate-buffered saline once for 3 min and fixed with CytoRich Red (Becton-Dickinson, Franklin Lakes, NJ, USA) at room temperature for 30 min.The fixed cells were washed with distilled water once for 3 min, deposited on a slide, further fixed in 95% ethanol, and stained with the above kit.At least 600 cells from three different fields of view were examined from each experiment.Cell death was expressed as the percentage of TUNEL-positive cells.

Tissue Samples
After obtaining approval from our institutional review board, we searched the surgical pathology database of our institution for the period July 1984 to June 2012.As shown in Table 1, we examined 42 cases of liposarcoma (14 atypical lipomatous tumors, six dedifferentiated liposarcomas, 21 myxoid liposarcomas, and one pleomorphic liposarcoma), 34 cases of lipoma and 20 cases of normal adipose tissue.The liposarcoma specimens included 27 primary tumors and 15 local recurrent or distant metastatic tumors.Only myxoid liposarcoma specimens included 10 primary, 10 local recurrent and 1 distant metastatic tumors.Diagnoses for all of these tumors were made according to the latest edition of the World Health Organization classification scheme (Fletcher et al., 2013).In regard to myxoid liposarcoma, fluorescence in situ hybridization analysis of the DNA damage-inducible transcript 3 (DDIT3) and fused in sarcoma (FUS) loci was performed in three of the 21 examined cases.We found that 3/3 cases showed both DDIT3 and FUS gene rearrangements.Clinical details and follow-up information were obtained by reviewing medical charts.The clinicopathologic characteristics of myxoid liposarcoma are summarized in Table 2.The 21 cases included 11 men and 10 women with an age range of 28-83 years (median, 52 years).Tumors were located in the extremities for 15 cases (thigh, 11; lower leg, three; forearm, one), in the trunk wall in five cases (buttock, three; abdominal wall, two), and in the retroperitoneum in one case.All primary tumors were treated with surgery, accompanied by adjuvant chemotherapy or irradiation in eight cases.For staging of the tumors, the latest American Joint Committee on Cancer (AJCC) staging system was used (Edge et al., 2010).The median follow-up period after surgery was 79 months (range, 5-244 months).Recurrence-free survival was defined as the interval from diagnosis to discovery of a local recurrence or the last follow-up examination.

Statistical Analysis
A t-test was used for comparison of means between two independent samples.Levene test was applied to verify the homoscedasticity.The univariate relationship between each clinicopathological variable and syndecan-1 expression was evaluated using the chi-squared test or Fisher's exact test.Recurrence-free survival was analyzed for 21 myxoid liposarcoma patients.Recurrence-free survival was estimated by the Kaplan-Meier method.The log-rank test was used to evaluate the differences between survival curves.These analyses were performed with IBM SPSS ver.20.0 (IBM Co., Armonk, NY, USA).A p-value less than 0.05 was considered statistically significant for all tests.

Syndecan-1 Immunostaining and Prognostic Significance
As a pilot study, we previously examined syndecan-1 expression by immunohistochemistry in 158 soft tissue sarcomas and found significant results in lipomatous tumors.Syndecan-1 expression was observed in 12% of all liposarcomas but not in lipomas and normal adipose tissue (Table 1).The results of MDM2 and p16 immunostaining are also shown in Table 1.No significant association between syndecan-1 expression and prognosis in each subtype of liposarcoma was detected (data not shown), except in myxoid liposarcomas.Therefore, we summarized the correlation between syndecan-1 immunopositivity and myxoid liposarcoma clinicopathologic factors in Table 2. Immunohistochemistry of myxoid liposarcoma showed that syndecan-1 was expressed in both the nucleus and the cell membrane (Figure 1).
A B Figure 1.Myxoid liposarcoma.Immunohistochemical analysis showed that syndecan-1 is expressed in both the nucleus and the cell membrane (A, ×100; B, ×400).
The two cases of syndecan-1 positive myxoid liposarcoma were primary and local recurrent tumors.Positive syndecan-1 staining showed a significant association with unfavorable recurrence-free survival of myxoid liposarcoma patients by a univariate prognostic analysis (p = 0.023; Table 3; Figure 2).Male was also associated with poor prognosis (p = 0.04; Table 3).We also investigated the relationships between percentage of syndecan-1 positive tumor cells and overall or metastatic-free survival in myxoid liposarcoma patients using the Kaplan-Meier method.We found no significant associations between percentage of syndecan-1 positive tumor cells and outcomes (data not shown).

Discussion
In our study, prognostic analyses showed that syndecan-1 expression might be an unfavorable prognostic factor for recurrence-free survival in myxoid liposarcoma patients (P < 0.05).However, owing to the patient numbers, statistical power is weak and thus is a limitation of this study.Previous studies on the roles of syndecan-1 during neoplastic transformation and progression of various human malignancies revealed various findings dependent on tumor type (Sanderson & Borset, 2002).In squamous cell carcinomas of the head and neck (Inki et al., 1994;Anttonen et al., 1999), colorectal adenocarcinoma (Day et al., 1999), breast carcinoma (Loussouarn et al., 2008), renal cell carcinoma (Gökden et al., 2006), hepatocellular carcinoma (Matsumoto et al., 1997) and malignant mesothelioma (Kumar-Singh et al., 1998), downregulation of syndecan-1 expression was observed with tumor progression.In contrast, increased syndecan-1 expression was seen in pancreatic and prostate adenocarcinomas compared with their benign counterparts (Conejo et al., 2000;Zellweger et al., 2003).Thus, syndecan-1 might have varying effects on the progression of different tumor types.Limited data are available regarding the prognostic value of syndecan-1 expression in soft tissue sarcomas (O'Connell et al., 2004;Orosz et al., 2001).
Our results suggest that syndecan-1 immunohistochemistry may help us to predict the risk of local recurrence of myxoid liposarcomas prior to clinical treatment.
In the present study, syndecan-1 knockdown suppressed cell growth through the induction of apoptosis in human liposarcoma cells.Syndecan-1 gene silencing induced growth arrest and apoptosis in myeloma cells.This could be because of diminished pro-survival signals as a result of reduced levels of cell surface heparan sulfate proteoglycan co-receptors for growth factor signaling (Khotskaya et al., 2009).This mechanism also applies to liposarcoma cells.Moreover, syndecan-1 synergizes with heparanase to promote myeloma bone disease by activating the HGF-Met-IL-11-RANKL signaling pathway (Teng et al., 2012).Therapy targeting the syndecan-1/heparanase axis can block the aggressive behavior of tumors, and could lead to new tumor therapies against drug-resistant cancers or sarcomas.Thus, our findings suggest that a therapy directly targeting syndecan-1 and heparanase inhibitors should be effective against myxoid liposarcomas.
In summary, our results show that syndecan-1 overexpression may be associated with unfavorable recurrence-free survival in myxoid liposarcoma patients.In addition, syndecan-1 may be essential for cell growth in liposarcoma in vitro.Therefore, syndecan-1 could represent a new molecular target and a useful clinicopathological marker in myxoid liposarcoma.

Figure 2 .
Figure 2. Kaplan-Meier analysis of recurrence-free survival in myxoid liposarcoma patients with syndecan-1 expression

Figure 3 .
Figure 3. Downregulation of syndecan-1 induced apoptosis in human liposarcomaA, SW872 cells were transfected with 132 nM syndecan-1 siRNA or control RNA.Cells were cultured for a further 48 h after transfection, and expression of syndecan-1 mRNA was determined by RT-PCR.B, SW872 cells were cultivated for 72 h after control RNA or syndecan-1 siRNA transfection and cell growth rates were determined.C, SW872 cells were cultivated for 72 h after control RNA or syndecan-1 siRNA transfection and

Table 2 .
Association between clinicopathologic variables and syndecan-1 expression in myxoid liposarcoma

Table 3 .
Univarite analysis for Recurrence-Free Survival in myxoid liposarcoma patients