Genetically, liposarcomas fall into two broad categories. Those with simple near-diploid karyotypes bear few chromosomal rearrangements, such as translocations in myxoid/round-cell liposarcoma [t(12;16)(q13;p11), t(12;22)(q13;q12)]. In contrast, liposarcomas with complex karyotypes, including well-differentiated/dedifferentiated and pleomorphic liposarcoma, have varying degrees of chromosomal instability (21
). Most of these complex liposarcoma types have frequent abnormalities in the Rb, p53, and specific growth-factor signaling pathways, including amplification of the 12q14 chromosome region encompassing the MDM2
loci in dedifferentiated liposarcoma (22
), but many of the key driver genes critical to liposarcomagenesis remain to be discovered. Here we show that ZIC1
is significantly overexpressed in all 5 liposarcoma subtypes compared with normal fat and that both genetically simple and complex liposarcoma types depend on ZIC1 overexpression for proliferation, invasion, and survival. We also discovered ZIC1 overexpression in other types of soft tissue sarcoma, including leiomyosarcoma, MFH, and myxofibrosarcoma. GIST is the only sarcoma type examined that did not overexpress ZIC1. As GIST tumorigenesis is primarily characterized by mutations in c-KIT
, lesions not characteristic of ZIC1-overexpressing subtypes, it is likely the pathogenesis of the latter is distinct in the requirement for ZIC1 overexpression (23
). The overexpression of ZIC1 in eight of nine soft tissue sarcoma and liposarcoma subtypes studied here make this gene a promising biomarker associated with mesenchymal transformation, and a potential selective therapeutic target due to the low or absent ZIC1 expression in adult tissues outside of the CNS.
In the CNS ZIC1 inhibits premature neuronal differentiation and increases cell proliferation in neural development. However, the underlying molecular actions of ZIC1 protein remain a mystery. Some studies have raised the possibility that ZIC proteins can act as transcriptional cofactors and modulate the hedgehog-signaling pathway and that ZIC proteins inhibit neuronal differentiation by activating Notch signals (5
). While the role of ZIC1 in the CNS has been investigated, the functional effects of ZIC1 overexpression in soft tissue sarcoma have, until now, not been examined.
Three different liposarcoma cell lines were used to model the potential proliferative role of ZIC1 overexpression in the liposarcoma human tumors. The functional effects of ZIC1 knockdown were consistent: decreases in proliferation, BrdU uptake, and Matrigel invasion and increase in apoptosis. Specificity of these responses are supported by ZIC1 knockdown having no effect on apoptosis or invasion in normal ASCs, which are neither immortal nor transformed, or in the A549 cell line, which does not to overexpress ZIC1 by RT-PCR and immunofluorescence. Thus, the overexpression of ZIC1 is essential for survival and proliferation in these liposarcoma cell lines and not in the normal progenitors or in proliferating cells in general.
The majority of ZIC1 studies have investigated its role in the CNS, and as previously mentioned, murine ZIC1 mutants were shown to express elevated p27 and Wnt7a, but decreased cyclin D1 (6
). While we did not observe a correlation between ZIC1 expression and Wnt7A and/or Cyclin D1, we did demonstrate a relationship between ZIC1 knockdown and an increase in p27 protein expression in vitro. As previously reported, increases in p27 expression have been shown to reduce proliferation (26
), reduce BrdU uptake(27
), increase Annexin V staining (28
), and decrease Matrigel invasion (29
). Immunoblots demonstrated an increase in p27 protein expression in the ZIC1 knockdowns as early as 2 days after lentivirus infection and reached its maximal level 6 days postinfection. In addition, immunofluorescence showed that ZIC1 knockdown also appeared to activate p27 by its nuclear localization. Thus, the increase in p27 expression in all 3 liposarcoma cell lines following ZIC1 knockdown explains many of the observed early functional effects on cell proliferation and invasion. However, the observed maximal increases in p27 by 6 days does not account for marked increase in cell death observed following 8 days of ZIC1 knockdown suggesting that additional ZIC1 target genes may be mediating liposarcoma cell survival.
To discover putative ZIC1
target genes we then performed expression array analysis 4 and 8 days following ZIC1
knockdown and found that BCL2L13 was 4-fold and 2-fold downregulated in DDLS and LPS141, respectively. BCL2L13 shows overall structural homology to the anti-apoptotic Bcl-2 family of proteins, but contains a C-terminal membrane anchor region that is preceded by a unique 250-amino acid insertion containing 2 tandem repeats. BCL2L13 induces significant apoptosis with caspase-3 activation when transfected into 293T cells (30
). BCL2L13 overexpression has been associated with L-asparaginase resistance and unfavorable clinical outcome in childhood acute lymphoblastic leukemia (31
). This finding suggests that BCL2L13 may have a different apoptotic role in primary leukemic cells and in our dedifferentiated liposarcoma cell lines compared with the 293T cell lines, initially used to describe its apoptotic role. Four days following ZIC1
knockdown JunD was 2.4-fold and 2.1-fold downregulated in DDLS8817 and LPS141, respectively. The JunD proto-oncogene is a functional component of the AP1 transcription factor complex and exhibits a cell-dependent role in apoptosis. For example, JunD expression prevents cell death in adult mouse heart cells (32
) and in UV/H2O2-stressed mouse embryonic fibroblasts (33
). JunD has been proposed to protect cells from p53-dependent senescence and apoptosis (34
) and its downregulation upon ZIC1
knockdown may account for the induction of apoptosis in our liposarcoma cell lines. Fam57A (CT120), a membrane-associated gene important for amino acid transport (35
), was found to be 2.6- and 2.5-fold downregulated in DDLS8817 and LPS141, respectively. CT120 has been implicated in lung carcinogenesis and its ectopic expression in NIH3T3 cells has been associated with Raf/MEK/Erk and PI3K/Akt signaling pathways driving cell proliferation, cell survival, and anti-apoptotic pathways (36
). EIF3M, eukaryotic initiation factor 3 subunit M, is an essential gene for global cellular protein synthesis and polysome formation and appears to be associated with the bulk of cellular mRNAs (37
). The ectopic expression of the other EIF3 subunits, −3a, −3b, −3c, −3h, or −3i in stably transfected NIH3T3 cells has been shown to induce oncogenic transformation, enhance proliferation, and attenuate apoptosis (38
). These findings suggest that the 3-fold downregulation of EIF3M following ZIC1
knockdown would inhibit protein synthesis and proliferation in liposarcoma cells.
We present here evidence that ZIC1 expression is necessary for liposarcoma proliferation, invasion, and survival and have identified several ZIC1 target genes that may mediate its functional effects. The overexpression of ZIC1 in both liposarcomas and, more broadly, other soft tissue sarcoma types and not in normal adult tissues outside of the CNS, makes this gene or its downstream targets a promising therapeutic target in these tumor types.