STAT3 is activated by a number of cytokines and growth factors and has diverse functions during embryogenesis and early development [25
]. Due to the transient nature of cytokine and growth factor signaling and the presence of proteins such as SOCS, s
ignaling, and PIAS, p
nhibitor of a
TAT, that inhibit of STAT3 signaling, STAT3 activation under normal physiological conditions is transient, lasting anywhere from several minutes to several hours [49
]. However, during tumorigenesis there is often constitutive activation of PTKs due to activating mutations or aberrant growth factor or cytokine signaling. Constitutive PTK signaling results in constitutive activation of STAT3[38
]. It is therefore not surprising that recent studies show that there is persistent activation of STAT3 in a number of human cancers including multiple myelomas, breast, ovarian and prostate carcinomas and head and neck tumors [32
The initial evidence that STAT3 signaling is critical to tumorigenesis came from the observation that activation of STAT3 is necessary for v-src induced transformation of NIH3T3 cells [38
]. Subsequent studies have shown that STAT3 activation is crucial to cellular transformation induced by a number of viral oncogenes [37
]. Inhibition of STAT3 signaling with either dominant negative or antisense oligonucleotides against STAT3 suppresses the transformation process in some tumors [34
]. Overexpression of a constitutively active mutant of STAT3 transforms 3T3 cells in culture [51
]. Finally, recent studies have shown that treatment of tumor cells with inhibitors of STAT signaling results in decreased cell viability and induces apoptosis [40
]. Together these findings demonstrate that STAT3 signaling plays a critical role in both the transformation process and tumor progression in some types of cancer.
Ourselves and others have found STAT3 is constitutively activated in astrocytomas and astrocytoma cell lines [41
]. This is consistent with the fact that human astrocytomas are often characterized by an amplification of either growth factor genes, such as PDGF, growth factor receptor genes, such as the EGF receptor, or cytokines such as IL-6 that can lead to constitutive activation of STAT3 [53
]. Not surprisingly, there was constitutive STAT3 activation in all astrocytoma cell lines tested. In contrast, NHA showed relatively little STAT3 expression and activation.
In order to determine the role of STAT3 in astrocytomas directly, we have used RNAi to specifically knock down the expression of STAT3 in several human astrocytomas. Treatment of astrocytoma cell lines with STAT3 siRNA resulted in morphologic and biochemical changes indicative of apoptosis. These results are consistent with results previously observed with dominant negative STAT3 in U251 cells [41
]. Although STAT3 siRNA decreased the amount of STAT3 present in NHA, it did not have a significant effect on their viability. This suggests that STAT3 may be an ideal target for cancer therapy since inhibition of STAT3 signaling induces tumor cell death, but does not kill normal astrocytes.
One mechanism by which STAT3 participates in tumorigenesis is by inhibiting apoptosis through the induction of anti-apoptotic genes. Biochemical studies have shown that astrocytoma cells express a number of death receptors but are resistant to death receptor mediated apoptosis [55
]. Other studies have shown that survivin is overexpressed in some tumors and that overexpression of the gene is sufficient to block apoptosis in tumor cells [48
]. We have found that both Bcl-xL, a member of the BCL2 family of proteins, and survivin are expressed in A172 cells (Figure &). Treating A172 cells with STAT3 siRNA significantly reduces expression levels of both of these genes. STAT3 responsive elements are found in the promoter region of both of these genes, suggesting that they are directly regulated by STAT3 [32
]. Our findings that Bcl-xL is regulated by STAT3 in astrocytoma cell lines is consistent with previous data [41
]. We are the first to report that STAT3 also regulates survivin expression in astrocytoma. Taken together with our data that knockdown of STAT3 can induce apoptosis, these findings suggest that induction of Bcl-xL and survivin genes by constitutively activated STAT3 promotes survival of astrocytoma cells.
Not all the astrocytoma cell lines examined responded equally to STAT3 knockdown. This suggests that some astrocytomas are not dependent on STAT3 for survival. Further work is needed to determine whether this STAT3 independence has evolved from STAT3 dependent cells or more likely is a result of the specific types of oncogenic mutations in these tumors.
Data presented in this paper are consistent with the growing body of evidence suggesting STAT3 may be an important therapeutic target in tumors including astrocytoma. We are the first to report that an siRNA can induce apoptosis of astrocytoma cells. siRNAs are short oligonucleotides of 21–23 nucleotides in length that can be used in vitro
to produce sequence specific gene silencing of mammalian cells [59
]. It has been shown that siRNAs can be used effectively in vivo
to suppress gene expression in adult mice [60
]. siRNAs can be directly introduced into the CNS to reduce endogenous gene expression [62
]. These results suggest that siRNA may become a useful clinical tool in the future. Since STAT3 signaling is important for the survival of a number of human tumors, STAT3 siRNA could become an effective therapeutic agent for STAT3 dependent tumors.