Oncogenesis is a discontinuous, progressive transformation of cells into a malignant and metastatic cell population. Tumor development depends on alterations in the genome and aberrant proliferation owing to autonomous growth-promoting signals, unresponsiveness to extracellular and intracellular growth-inhibitory signals, and evasion of apoptosis. Subsequent progression of the cancer into a malignant and metastatic phenotype relies on unrestrained replicative potential, avoidance of immune surveillance, stimulation of angiogenesis, inappropriate tissue invasion, and metastasis. Coverage of all intracellular signaling pathways associated with oncogenesis is beyond the scope of this chapter, and we focus instead predominantly on the roles of the Jak-Stat pathway and selected members of the Src family protein tyrosine kinases as well as the SOCS proteins.
Although Stat transcription factors have not been implicated in DNA damage detection and repair systems, numerous reports outline their involvement in cell-cycle progression, apoptosis, and cytokine-induced survival, proliferation, and growth inhibition (69-74). Indeed, numerous data show the presence of activated Stat proteins in a variety of cancers (reviewed in refs. 75 and 76). Numerous studies indicate that activation of Jak-Stat pathways is required for transformation by the oncoprotein, v-src, which drives expression of c-myc, an important mediator of cell proliferation and differentiation (77-82). It is important, however, to distinguish between a primary role for these proteins in disease progression and secondary consequences of tumorigene-sis. To this end, it should be established both in cell lines and in animal models that activation or removal of a component of the signaling pathway, such as the Jak-Stat pathway, is essential or sufficient for cellular transformation or oncogenesis. The growth-suppressive functions of Stat1 indicate that it may be a potent tumor suppressor, evidenced by an increase in susceptibility to tumors in murine Stat1 knockout models. Numerous studies in cell lines and in vivo have shown the oncogenic potential of Jak1, Jak2, Jak3, Stat3, and Stat5. Circumstantial evidence in mouse models and methylation and expression patterns have indicated that SOCS proteins may also act as tumor-suppressor proteins.
Growth of tumors such as childhood acute lymphoblastic leukemia (ALL) is often dependent on cytokine stimulation, presumably through a paracrine or autocrine pathway, which induces phosphorylation and activation of receptors and associated signaling pathways, including the Jak-Stat pathway. Specific inhibition of Jak2 in ALL and acute myeloid leukemia (AML) by the tyrphostin AG490, a specific tyrosine kinase blocker, inhibited proliferation and induced apoptosis of leukemic cells without significant perturbations in hematopoiesis (83,84). Another form of ALL is associated with a chromosomal translocation fusing the catalytic kinase domain of Jak2 with an ETS transcription factor family member TEL, also known as ETV6. TEL is characterized by a DNA-binding domain (conserved in ETS family members) and importantly, a helix-loop-helix oligomerization domain that allows TEL-Jak2 to dimerize and activate Jak2 kinase activity, thereby activating signaling pathways. Other common cytogenetic rearrangements resulting in neoplastic growth include TEL-ABL and TEL-AML fusions (85-88). The TEL-Jak2 and TEL-ABL, but not BCR-ABL fusion proteins induced Stat3 and Stat5 activation and cytokine-independent growth of an IL-3-depen-dent BaF3 hematopoietic cell line (84,89,90).
The growth-suppressive and proapoptotic activities of Stat1 in response to cytokines such as IFN-y, TNF-a, and IL-6 are well documented. In the absence of Stat1, deficiencies occur in constitutive expression of caspases and IFN-y-induced expression of cas-pase 1 and the cyclin-dependent kinase inhibitor p21WAF1/CIP1 leading to an impairment of cell growth arrest and apoptosis (91-94). An important step in tumor development is evasion of the immune system, in which IFN-y plays an important role by upregulating expression of MHC proteins for antigen presentation. As described earlier, Stat1-
deficient mice are unresponsive to both type I and II IFN, and mice deficient in Stat1 or the IFN-y receptor are more susceptible to spontaneous or chemical carcinogen-induced tumor development. The progression of tumor development is greater in mice deficient in both p53 and the IFN-yR than for p53-deficient mice, and the variety of tumors is more diverse (95). Recent evidence indicates that IFN-y can support effects on cell proliferation and induce an antiviral state independently of Stat1, suggesting that results obtained from mice deficient in IFN-y may not necessarily be equivalent to those simply deficient in Stat1 (96,97).
Extensive data describe activated Stat3 and Stat5 in tumors. Activating mutations of Stat3 and Stat5 and specific inhibition of Stat3 and Stat5 by gene deletion, antisense oligonucleotides, or dominant negative approaches have highlighted the importance of Stat3 and Stat5 in tumor formation. Mutations of Stat3 that allow spontaneous dimer-ization of the monomers in the absence of interactions between phosphorylated tyrosines and SH2 domains are sufficient to cause transformation and induce tumor formation in nude mice (98). Inhibition of Stat3 signaling using antisense Stat3 or by a Jak-selective tyrosine kinase inhibitor, AG490, restored the sensitivity of cells from patients with large granular lymphocyte (LGL) leukemia to Fas-mediated apoptosis (99). Downregulation of Fas correlates with an increase in metastatic potential and resistance of tumors to chemically and physically induced apoptosis. This effect is mediated, at least in part, by an interaction between Stat3 and c-jun, which decreases expression of Fas (100). Fas expression is increased in mice with a heterozygous disruption of Stat3 or a homozygous deletion of c-jun. Furthermore, mutation of promoter binding sites for c-jun (AP-1) and Stat3 (GAS) increases Fas expression. Expression of dominant negative Stat3 increases Fas in a mouse melanoma model, decreases proliferation in a breast carcinoma model, and promotes apoptosis in a human myeloma cell line by inhibiting expression of the antiapoptotic protein Bcl-xL (100-102). Inhibition of Stat3 using antisense Stat3 oligonucleotides inhibits proliferation and induces apoptosis in human prostate cancer cells and squamous cell carcinoma cell lines (103,104). Inhibition of Stat3 in a murine head and neck xenograft tumor model using liposome-delivered plasmid antisense STAT3 gene therapy inhibited proliferation and induced apoptosis in the tumor by downregulating expression of the antiapoptotic protein Bcl-xL (105). An activating mutation at Asp8i6 of c-kit, a receptor tyrosine kinase for SCF, is associated with numerous malignancies and is characterized by constitutive activation of Stat3. Overexpression of dominant negative Stat3 inhibited anchorage-independent growth of mutant c-kit cell lines and tumor formation in NOD/SCID mice (106).
In these studies in which Stat3 function is inhibited by various approaches, it is assumed that the inhibition of proliferation and induction of apoptosis is directly linked to an absence of Stat3 function, and investigators have speculated on techniques of intervention in tumor formation by specific inhibition of Stat3. It was shown that Stat3-deficient, primary embryonic fibroblasts stimulated with IL-6 produce an IFN-y-like response by activation of Stat1 and upregulation of IFN-y-inducible genes, producing an antiviral response in the presence or absence of IFN-y (107). Stat3 conditional knockout mice display increased Stat1 activation in bone marrow (21). It is unclear by what mechanism Stat3 provides specificity for IL-6 signaling and prevents activation of an IFN-y-like Stat1-mediated response, but it has profound implications for the potential use of specific Stat3 inhibitors in the clinic.
Like Stat3, Stat5 also plays a fundamental role in regulation of proliferation, differentiation, and apoptosis of hematopoietic cells (108) by coordinating the expression of SOCS proteins as well as antiapoptotic genes such as bcl-xL and pim-1 and growth-inhibitory genes such as p21WAF1/Cip1. The TEL/Jak2 fusion protein, found in ALL and chronic myelogenous leukemia (CML), induced a myelo- and lymphoproliferative disease when TEL/Jak2 transformed bone marrow cells were transplanted into ablated recipients but had no effect on a Stat5a/b null background. Stat5a/b was not required for TEL/Jak2-induced growth factor-independent murine bone marrow-colony formation (109). Importantly, expression of a constitutively active version of Stat5a in bone marrow recapitulated the phenotype of bone marrow transformed by TEL/Jak2. In acute promyelocytic leukemia (APL), a Stat5b-retinoic acid receptor a (RARa) fusion protein has been identified. RARa is a member of the nuclear hormone receptor family and serves to regulate differentiation and growth inhibition (110). In this model, the Stat5b-RARa fusion protein altered transcription of target genes by binding retinoic acid response elements (RAREs) as a homodimer or as a heterodimer with the retinoid X receptor a (RXRa). In CML, BCR-ABL activates Stat5 through interactions with Jak (111). Using a dominant negative approach, Stat5 activation was shown to be essential for proliferation, evasion of apoptosis, and tumor progression in a BCR-ABL-dependent model for leukemia in mice (112). Overexpression of dominant negative Stat5a suppresses BCR-ABL-driven proliferation of Ba/F3 cells (113). In the WAP-Tag transgenic mouse model for mammary cancer, 86% of the adenocarcinomas display activated Stat5a. Loss of one allele of Stat5a in transgenic mice reduced tumor incidence, reduced the size and time of first onset of tumors, and increased the apop-totic index of mammary cancer cells (114). In mammary carcinoma cells, PRL induces proliferation by inducing cyclin D1 expression through a Jak2/Stat5 signaling pathway (115).
Several studies indicate that insufficiency in negative regulation of Jak-Stat pathways by the SOCS proteins could contribute to oncogenesis (116,117). SOCS1 inhibits transformation by TEL-Jak2, an activated c-KIT receptor, v-ABL and to a lesser degree BCR-ABL but not transformation induced by TEL-ABL, v-SRC, or RasV12 (118-120). Mutation of the SH2 domain prevented SOCS1 inhibition of TEL-Jak2-induced cellular transformation, but not that of the mutant KIT receptor, indicating that more than one mechanism of tumor-suppressor activity exists for SOCS1. SOCS1 overexpression with TEL-Jak2 can prolong the latency period of murine bone marrow transplants compared with TEL-Jak2 alone. One mechanism for negative regulation of TEL-Jak2-mediated cellular proliferation is the SOCS-box dependent proteasomal degradation of TEL-Jak2 (121). Importantly, SOCS1-defi-cient fibroblasts are more sensitive to transformation induced by TEL-Jak2 or activated KIT. Other studies have shown that the presence of SOCS1 does not necessarily inhibit the oncogenic activity of TEL-Jak2, which bypasses the actions of SOCS1 (122). Similarly, constitutive expression of SOCS1 and SOCS3 in AML or cutaneous T-cell lymphoma may reflect the constitutive activation of cytokine-signaling pathways rather than reflecting inhibition of autocrine or paracrine cytokine-induced Stat3 activation (123,124). In some human primary tumors, including human hepatocellular carcinoma and multiple myeloma, silencing of the SOCS-1 gene by hypermethylation and loss of heterozygosity suggest a direct tumor suppressor role of SOCS-1 (117,125-127).
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