Retroviruses have played profound roles in our understanding of the genetic and molecular basis of cancer. Jaagsiekte sheep retrovirus (JSRV) is a simple retrovirus that causes contagious lung tumors in sheep, known as ovine pulmonary adenocarcinoma (OPA). Intriguingly, OPA resembles pulmonary adenocarcinoma in humans, and may provide a model for this frequent human cancer. Distinct from the classical mechanisms of retroviral oncogenesis by insertional activation of or virus capture of host oncogenes, the native envelope (Env) structural protein of JSRV is itself the active oncogene. A major pathway for Env transformation involves interaction of the Env cytoplasmic tail with as yet unidentified cellular adaptor(s), leading to the activation of PI3K/Akt and MAPK signaling cascades. Another potential mechanism involves the cell-entry receptor for JSRV, Hyaluronidase 2 (Hyal2), and the RON receptor tyrosine kinase, but the exact roles of these proteins in JSRV Env transformation remain to be better understood. Recently, a mouse model of lung cancer induced by JSRV Env has been developed, and the tumors in mice resemble those seen in sheep infected with JSRV and in humans. In this review, we summarize recent progress in our understanding the molecular mechanisms of oncogenic transformation by JSRV Env protein, and discuss the relevance to human lung cancer.
Sheep retroviral oncogenesis: a historical perspective
For almost a hundred years, retroviruses have been known to induce tumors. The field started with the discovery of avian erythroblastosis virus and Rous sarcoma virus, both of which cause tumors in chickens (for a comprehensive review see Rosenberg and Jolicoeur (1997)). Oncogenic retroviruses are historically classified into acute transforming retroviruses and nonacute retroviruses. Acute transforming retroviruses induce tumors through acquisition and expression of cellular proto-oncogenes – a process referred to as oncogene capture. These retroviruses induce tumors rapidly, the tumors are of polyclonal origin, and the viral oncogenes in these viruses are not essential for virus replication. In fact, acute transforming retroviruses are often replication-defective (Rous sarcoma virus is an exception) because of the loss or disruption of essential viral genes during the oncogene capture process. Nonacute retroviruses, on the other hand, do not carry oncogenes and induce tumors slowly. This type of retrovirus causes tumors by activating cellular proto-oncogenes close to the proviral DNA integration sites – a process termed insertional activation. Numerous replication-competent retroviruses cause clonal tumors by proviral insertional activation. In addition to oncogene capture and insertional activation, a few replication-competent retroviruses induce tumors by expression of viral proteins. For example, human T-cells leukemia virus types 1 and 2 (HTLV-1 and HTLV-2, respectively) induce adult T-cell immortalization and leukemia in humans by expression of viral Tax proteins that transactivate several cytokines and cytokine receptors. It is worth noting that HTLV-1 and HTLV-2 are the only retroviruses identified so far, that directly cause human cancer.
Jaagsiekte sheep retrovirus (JSRV) is simple retrovirus encoding Gag, Pro, Pol, and Env proteins (York et al., 1992; Palmarini et al., 1999; DeMartini et al., 2001), and is now classified as an ovine betaretrovirus. This family also includes a closely related retrovirus of sheep and goats, enzootic nasal tumor virus (ENTV) (De las Heras et al., 2003). JSRV transforms peripheral lung epithelial cells leading to ovine pulmonary adenocarcinoma (OPA) (Palmarini et al., 1999; DeMartini et al., 2001), while ENTV transforms nasal epithelial cells resulting in enzootic nasal tumor (ENT) (De las Heras et al., 2003). OPA is a significant veterinary and economic problem in many countries, and in some countries such as Britain and South Africa, the incidence of OPA can be as high as 30% (Sharp and Angus, 1990). The clinical signs of OPA are associated with respiratory dysfunction and include exaggerated breathing after exercise. Late stages of disease are often accompanied by the secretion of copious lung fluid that contains infectious virus.
JSRV can induce lung tumors in newborn lambs in as little as 10 days following experimental inoculation (Verwoerd et al., 1980), showing that JSRV is an acutely transforming retrovirus. However, JSRV is a simple retrovirus with no apparent oncogene present in its genome (Figure 1a). An alternate open reading frame, orf-X, which is present within the pol region of JSRV, was initially suspected to be an oncogenic factor but was later excluded. Orf-X bears no resemblance to any known cellular oncogenes except sharing a weak similarity with the adenosine A3 receptor (Bai et al., 1999). Disruption of orf-X has no effect on cell transformation by JSRV (Maeda et al., 2001). Remarkably, expression of native JSRV Env protein alone is sufficient to induce cell transformation in culture (Maeda et al., 2001; Rai et al., 2001; Allen et al., 2002; Danilkovitch-Miagkova et al., 2003; Liu and Miller, 2005), indicating that Env is an active oncogene and is likely to be the key factor in sheep oncogenesis. The only other examples of retroviruses with oncogenic Env proteins are ENTV (Alberti et al., 2002; Dirks et al., 2002), a close relative of JSRV; avian hemangioma virus, which expresses an Env that induces proliferation of monkey epithelial cells and NIH 3T3 cells (Alian et al., 2000); and spleen focus-forming virus, a replication-defective virus that expresses a recombinant nonfunctional Env protein that induces cell proliferation in cultured cells and in animals (Ruscetti, 1999). Therefore, JSRV and ENTV are members of a small family of acutely transforming retroviruses in which an Env protein acts as an oncogene.
Intriguingly, lung tumors induced by JSRV in sheep morphologically resemble a subtype of human pulmonary adenocarcinomas previously referred to bronchiolo-alveolar carcinoma or BAC (Bonne, 1939). For example, both OPA and BAC tumor cells express markers of type II pneumocytes and Clara cells, and tumors are generally multifocal and localized to the periphery of the lungs (Mornex et al., 2003). However, given the new definition of BAC by WHO (Travis et al., 1999), important distinctions exist between OPA and BAC. BAC is now defined as a peripheral, well differentiated adenocarcinoma with pure bronchio-alveolar growth and no evidence of stromal, vascular and pleural invasion (Travis et al., 1999); while OPA is a mixed peripheral adenocarcinoma with acinar, papillary and bronchio-alveolar growth (Palmarini and Fan, 2001). While the true frequency of pure BAC remains uncertain, tumors with histologically mixed BAC and adenocarcinoma account for >20% all non-small cell lung carcinomas (NSCLC) (Raz et al., 2006), and are increasing in frequency (Barsky et al., 1994).
Similarities between OPA and human lung adenocarcinoma have led to the hypothesis that JSRV or a related virus might be involved in human cancer. In one study, antisera raised against the JSRV capsid protein recognized ∼30% of human BAC and lung adenocarcinoma samples but in general did not recognize other tumor types or normal lung tissue (De las Heras et al., 2000). In this case, PCR studies were not carried out to confirm the presence of a retrovirus related to JSRV. Others have attempted to identify JSRV-related sequences in human lung cancer specimens by PCR. In one case, orf-X-like and gag-pro-like sequences were detected in 10–26% of lung cancer patients and healthy individuals in Nigeria and Cameroon, but were not present in samples from France, Germany and Russia (Morozov et al., 2004). In two other studies, JSRV-like sequences were not detected (Yousem et al., 2001; Hiatt and Highsmith, 2002). Together these studies suggest the possibility of JSRV involvement in human lung cancers, but more work is needed to resolve this issue.
JSRV and ENTV cell-entry receptor Hyal2: a putative tumor suppressor
Entry of retroviruses into target cells is dependent on specific interactions between the Env proteins present on the virus surface and their corresponding cell-entry receptors on the cell surface. The human receptor for JSRV and ENTV has been identified as hyaluronidase 2 (Hyal2) by phenotypic screening of human/hamster radiation hybrid cell lines, based on the fact that JSRV vectors transduce human but not hamster cells (Rai et al., 2000, 2001). Interestingly, unlike most retroviral receptors or coreceptors which are single or multiple transmembrane proteins (Overbaugh et al., 2001), the JSRV/ENTV entry receptor Hyal2 is attached to cell surface by a glycosylphosphatidylinositol (GPI)-anchor (Rai et al., 2001). Hyal2 orthologs have been cloned from several other species, including sheep, mouse, rat, cow, pig and dog. Ovine Hyal2 functions best as a receptor for JSRV and ENTV, as might be expected (Dirks et al., 2002). When overexpressed, rat Hyal2 can function as a receptor for JSRV but not for ENTV (Liu et al., 2003a), whereas mouse Hyal2 is inactive as a receptor for either virus (Dirks et al., 2002; Liu et al., 2003a). The receptor activities of these Hyal2 orthologs are in accordance with the host range of JSRV infection; for instance, ovine and human cells are highly susceptible to JSRV vector transduction compared to rodent cells (Rai et al., 2000; Dirks et al., 2002). Domains or amino-acid residues that are responsible for these different receptor activities are complex, but appear to reside in the central third of the Hyal2 protein sequence (Duh et al., 2005).
Human Hyal2 is located in the p21.3 region of chromosome 3 (Rai et al., 2000), a region that is deleted in a substantial proportion of human cancers (Zabarovsky et al., 2002). Hyal2 and other genes in this region are therefore regarded as putative tumor suppressors (Zabarovsky et al., 2002). Hyal2 belongs to the hyaluronidase gene family that includes Hyal1, PH20, Hyal3 and Hyal4 (Csoka et al., 2001). Hyal2 exhibits low yet detectable hyaluronidase activity compared to that of Hyal1 and PH20 (Lepperdinger et al., 1998; Rai et al., 2001; Vigdorovich et al., 2005). Hyaluronidases degrade hyaluronan, a major component of extracellular matrix that is important for the maintenance of normal tissue morphology, cell–cell communication, and possibly in tumor metastasis (Menzel and Farr, 1998). The normal biologic function of Hyal2 is not known, but Hyal2 appears to play an important role in JSRV Env transformation of human airway epithelial cells (Danilkovitch-Miagkova et al., 2003) (see below). Other GPI-anchored proteins have been shown to be involved in cell signaling, for example, a 150-KDa GPI-anchored TGF-β1-binding protein was recently found to regulate TGF-β signaling in human keratinocytes (Tam et al., 2003).
JSRV Env is an oncogene: primary structure and oncogenic domains
The primary function of retroviral Env proteins is to mediate viral entry into host cells through binding to specific entry receptors on the cell surface. However, JSRV Env also transforms cultured cells. Given that diseased sheep often secrete a large amount of lung fluid containing infectious JSRV particles (Palmarini and Fan, 2001) and the fact that JSRV expression is solely detected in tumor cells but not in surrounding normal cells (Palmarini et al., 1995), it is postulated that cell transformation by JSRV Env may be advantageous for viral replication in vivo (Fan et al., 2003).
JSRV Env is a typical type-I transmembrane protein of approximately 620 amino acids in length before processing (York et al., 1992; Palmarini et al., 1999; DeMartini et al., 2001). The mature full-length Env is composed of surface (SU) and transmembrane (TM) subunits that are linked by disulfide bonds (Figure 1b). SU is responsible for receptor-binding, and TM is involved in virus–cell membrane fusion. As for other simple retroviruses, JSRV Env has a short cytoplasmic domain of ∼44 amino acids in length (Figure 1c). Interestingly, the cytoplasmic tail of JSRV Env harbors a YXXM (Y is tyrosine, X stands for any amino acid, M is methionine) peptide motif, a putative binding site for the PI3K/p85 regulatory subunit (Songyang et al., 1993). This feature is distinct from most other retroviruses that contain the YXXΦ motif (Φ stands for any amino acids with a bulky hydrophobic chain). The YXXΦ motif has been shown to play important roles in endocytosis, trafficking, as well as retroviral pathogenesis. The role of YXXM motif in JSRV Env transformation has been investigated, and is summarized below.
The Env cytoplasmic tail: necessary and sufficient for cell transformation?
Studies from several groups demonstrate that the cytoplasmic tail of JSRV Env is essential for transformation, because replacement of this domain with that of other retroviruses completely abolishes the transforming activity of JSRV Env in multiple cell lines, including NIH 3T3 mouse fibroblasts (Palmarini et al., 2001), 208F rat fibroblasts (Liu et al., 2003b), DF-1 chicken fibroblasts (Allen et al., 2002), and MDCK canine epithelial cells (Liu and Miller, 2005). However, whether or not this domain is sufficient for JSRV Env transformation still remains elusive. We and others have attempted to address this issue by building the cytoplasmic domain of JSRV Env into other retroviral envelope proteins, but in general these proteins fail to be expressed well and fail to transform. One study reported that the C-terminal 141-amino-acid, but not the 44 amino-acid cytoplasmic domain, of JSRV Env protein was able to efficiently transform 208F rat fibroblasts (Chow et al., 2003). This was accomplished by attaching the C-terminus of JSRV Env sequence to a myristoylation signal derived from the Rasheed sarcoma virus gag protein. However, we could only detect minimal if any transforming activity of this construct in 208F cells or other cell lines (unpublished results). Similar results were also obtained by other investigators, including this group (Varela et al., 2006). Thus, it is possible that the cytoplasmic tail of JSRV Env is necessary but not sufficient for cell transformation, but more work is required to resolve this issue.
Role of the YXXM motif in cell transformation
It has been hypothesized that the YXXM peptide motif present in the cytoplasmic tail of JSRV Env, a putative PI3K/p85 binding motif, might be essential for cell transformation, especially given its absolute conservation among all transforming JSRV and ENTV strains yet absence in the nononcogenic endogenous sheep retroviruses (Palmarini et al., 2000b). Indeed, an early study showed that mutation of this tyrosine residue to phenylalanine or to aspartic acid completely abolished the transforming activity of JSRV Env in NIH 3T3 cells (Palmarini et al., 2001). However, we and others later observed that all of these tyrosine mutants were still able to transform NIH 3T3 (Liu et al., 2003b), 208F (Liu et al., 2003b), DF-1 (Allen et al., 2002), and MDCK (Liu and Miller, 2005) cells, but with reduced efficiency in most cases. The difference in results is likely due to different transformation procedures used by each group, including differences in the duration of transformation assay and use of G418 selection after transfection. In addition, despite repeated attempts, no tyrosine phosphorylation has been detected for the JSRV Env protein in the transformed cells (Liu et al., 2003b), which argues against a direct role of the YXXM motif in activating the PI3K/Akt signaling pathway (see below). Furthermore, mutation of the methionine residue of the YXXM motif also failed to have significant effect on cell transformation (Allen et al., 2002; Liu et al., 2003b). Moreover, the YXXM Env mutant-transformed cells still show robust PI3K-dependent Akt activation (Liu et al., 2003b). Thus, it is now generally believed that the tyrosine residue of YXXM motif is important for Env transformation, possibly by modulating the Env protein expression or its overall configuration, but the YXXM motif apparently does not directly activate PI3K in transformed cells.
Is SU involved?
The role of SU subunit in JSRV Env cell transformation appears to depend on the target cell type, but exactly how it affects transformation in these cells is poorly understood. Several groups have investigated whether SU is important for rodent fibroblast transformation by JSRV Env, but have obtained inconsistent results. In one study, Chow et al. (2003) replaced the receptor-binding domain and proline-rich region of JSRV SU with that of Moloney murine leukemia virus, or deleted this receptor-binding domain from SU, but found no dramatic effect on Env transformation of 208F cells (Chow et al. 2003). This finding is consistent with our data that JSRV SU protein does not bind the mouse ortholog of Hyal2 receptor in NIH 3T3 cells, and that overexpression of mouse Hyal2 has no effect on JSRV Env transformation in NIH 3T3 and 208F cells (Liu et al., 2003a). By contrast, Hofacre and Fan (2004) recently showed that large deletions or small insertions in the JSRV SU region could abolish JSRV Env transformation of NIH 3T3 and 208F cells. Interestingly, these investigators observed that co-transfection of transformation-defective SU and TM mutants was able to rescue the Env transforming activity in 208F cells (Hofacre and Fan, 2004), suggesting that efficient transformation of rodent fibroblasts by JSRV Env may require both SU and TM.
While it is easy to understand the direct role of the cytoplasmic domain of JSRV Env in cell transformation, how SU subunit might be involved in this process remains unclear. One possibility is that SU transmits independent signals that synergize with those of the Env cytoplasmic domain, as proposed by Hofacre and Fan (2004). Alternatively, SU may contribute to cell transformation indirectly by modulating the overall structure of Env protein. In both cases, cellular proteins might be involved. Thus, it will be important to identify cellular adaptor molecules that directly interact with JSRV SU and/or TM subunits, and to investigate how these molecules mediate intracellular signaling, leading to cell transformation.
Unlike the situation in rodent fibroblasts, JSRV SU and its interaction with receptor Hyal2 appear to be critical for the Env transformation of BEAS-2B human bronchial epithelial cells. BEAS-2B is an immortalized cell line established by infection with a hybrid adenovirus-12 and SV40 virus (Reddel et al., 1988). Transfection of BEAS-2B cells with a plasmid encoding JSRV Env was shown to result in piled-up colonies with fork-like structures, indicative of transformation (Danilkovitch-Miagkova et al., 2003). Interestingly, Hyal2 and its interaction with JSRV Env seem to play a critical role in the Env transformation of this cell line. In normal BEAS-2B cells, Hyal2 was found in a complex with RON receptor tyrosine kinase and therefore inhibit RON activation, expression of JSRV Env resulted in Hyal2 proteasomal degradation, thereby liberating RON and leading to its activation (Danilkovitch-Miagkova et al., 2003). As the receptor-binding domain that interacts with Hyal2 resides in the SU subunit of Env protein (Liu et al., 2003a), the SU subunit is presumably responsible for the Env transformation of BEAS-2B cells. However, whether or not SU or the receptor-binding domain of SU is necessary and sufficient for BEAS-2B cell transformation, or if other regions of JSRV Env are also required in the transformation process, need to be addressed. In addition, these experiments have been difficult to reproduce because of a high background of transformed foci and the poor transfectability of BEAS-2B cells.
Signaling pathways involved in JSRV Env-mediated cell transformation
Three signaling pathways have been reported to be involved in cell transformation by JSRV Env protein (Figure 2). The first is Akt pathway that is either dependent or independent of PI3K. This pathway is activated in Env-transformed fibroblasts derived from rodent (NIH 3T3 and 208F) (Palmarini et al., 2001; Alberti et al., 2002; Chow et al., 2003; Maeda et al., 2003, 2005; Liu et al., 2003b) and chicken (CEF and DF-1) (Allen et al., 2002; Zavala et al., 2003), as well as in MDCK dog epithelial cells (Liu and Miller, 2005). The second is Raf-MEK-MAPK pathway, which is activated in Env-transformed NIH 3T3 mouse fibroblasts and RK3E rat epithelial cells (Maeda et al., 2005). The third is referred to as RON-Hyal2 pathway, which is primarily activated in the transformed BEAS-2B human epithelial cells (Danilkovitch-Miagkova et al., 2003). Studies from several labs have shown that multiple signaling pathways can be activated in the same cell types transformed by JSRV Env, and the same pathway can be operative in different transformed cell lines. Below we highlight evidence that support for the roles of each pathway in JSRV Env transformation, and discuss some unsolved problems.
PI3K-dependent and independent Akt pathways
The PI3K-dependent Akt signaling pathway is well known for its role in cell survival, oncogenic transformation and cancer development (Datta et al., 1999). This pathway can be activated by many growth factors, hormones, cytokines, as well as by mutations or overexpression of receptor or nonreceptor tyrosine kinases (Datta et al., 1999). Many viral proteins, including retroviral oncogenes, can also activate this pathway. Importantly, PI3K and Akt themselves were originally discovered from the acutely transforming retroviruses, avian sarcoma virus 16 (ASV16) (Chang et al., 1997) and AKT8 (Bellacosa et al., 1991), respectively. The vial counterparts of PI3K and Akt, v-p3K and v-Akt, are also potent oncogenes that transform cells in culture (Chang et al., 1997; Aoki et al., 1998). The PI3K-independent Akt activation, on the other hand, is less common with only limited study. In the latter case, Akt is activated by cAMP, an agonist of PKA pathway, and is directly phosphorylated by calcium/calmodulin dependent kinase (CaMKK), in a PI3K-independent of manner (Sable et al., 1997; Filippa et al., 1999).
Several lines of evidence indicate that PI3K-dependent Akt activation is critical for cell transformation by the JSRV Env protein. First, Akt is activated in several Env-transformed cell lines, as shown by Akt phosphorylation and elevated in vitro Akt kinase activity (Palmarini et al., 2001; Zavala et al., 2003; Liu et al., 2003b; Liu and Miller, 2005). Second, PI3K specific inhibitor LY294002 or wortmannin inhibits transformation by JSRV Env in these cells, and inhibits their Akt phosphorylation and in vitro kinase activity in a dose-dependent manner (Palmarini et al., 2001; Zavala et al., 2003; Liu et al., 2003b). Third, PI3K-specific inhibitors are able to reverse the transformed phenotypes of Env-transformed 208F cells (Liu et al., 2003b). In addition, there appears to be a correlation between the transformed phenotype and the extent of Akt phosphorylation in transformed cells, with highly transformed cells by JSRV Env exhibiting higher Akt phosphorylation (Liu et al., 2003b). Despite these important correlations, no interaction between JSRV Env protein and PI3K/p85 has been observed, indicating JSRV Env may activate PI3K/Akt indirectly, possibly through as-yet unidentified cellular adaptor molecules or by other signaling pathways that are directly triggered by JSRV Env. Interestingly, however, Akt phosphorylation was not detected in the lung sections of sheep OPA, nor in the JSRV Env-transformed DF-1 cells in vitro, yet was present in the ENTV-induced nasal tumors of sheep (Zavala et al., 2003). These data indicate that the Akt phosphorylation in the Env-transformed cells may be cell-type dependent.
PI3K-independent Akt activation has also been reported for JSRV Env transformation. Fan and co-workers co-transfected NIH 3T3 cells with plasmids encoding JSRV Env and a dominant-negative mutant of p85, or transfected the Env-coding plasmid to mouse embryo fibroblasts derived from p85α/p85β-double-knockout mice, and observed that Env transformation efficiency in these cells were not significantly affected (Maeda et al., 2003). Interestingly, however, Akt phosphorylation was still readily detected from the Env-transformed cells, suggesting a PI3K-independent Akt activation by JSRV Env. Further evidence supporting this scenario came from the fact that PI3K inhibitor LY294002 or wortmannin failed to specifically inhibit the Env-mediated transformation in NIH 3T3 cells and to reverse the transformed phenotypes (Maeda et al., 2003). These results are quite provocative, because they stand in contrast with other studies and with the notion that Akt activation involved in cell survival and transformation is generally PI3K-dependent.
While it is not currently clear what factors may have contributed to these discrepancies, several possibilities exist. First, the mechanism of JSRV Env transformation might be cell type-dependent, and this has been proven to be the case for several viral oncogenes. For example, transformation of NIH 3T3 cell by viral Src (v-Src) oncogene is dependent on cellular Ras (c-Ras) activation, whereas v-Src transformation of Rat-2 fibroblasts does not involve c-Ras (Aftab et al., 1997). Second, different experimental procedures used by each group, for example, the duration of drug treatment or the drug concentration, may account for the distinct results. Third, although p85α and p85β, two major regulatory units of PI3K, were knocked out from the NIH 3T3 cells in the study by Maeda et al. (2003), other PI3K regulatory subunits, such as p55γ, may still be expressed in these cells that could mediate a PI3K-dependent Akt activation. Future work should address if Akt activation is mediated by other PI3K regulatory subunits in the p85α and p85β double-knockout cells transformed by JSRV Env, and if the PI3K-independent Akt activation is operative in other Env-transformed cell lines.
While Akt is activated in rodent fibroblasts transformed by JSRV Env protein, either in a PI3K-dependent or independent fashion, the signaling molecules that directly interact with JSRV Env protein and initiate the signaling network are still not known. Attempts have been made by several groups to identify the Env-interacting cellular molecules without success. Recently, toll-like receptor 4 (TLR4) was found to interact with JSRV Env protein, but the relevance of this interaction to cell transformation remains uncertain (Hung Fan, personal communication).
Likewise, the downstream Akt effector targets involved in JSRV Env transformation are also poorly understood. Among the diverse array of phospho-Akt (pAkt) substrates that have been identified so far, mTOR, the mammalian target of rapamycin, was recently found to be involved in JSRV Env transformation of NIH 3T3 cells (Maeda et al., 2005). This was shown by the fact that mTOR inhibitor, rapamycin, partially inhibits the Env transformation efficiency (Maeda et al., 2005). However, to what extent the mTOR activation contributes to transformation of NIH 3T3 cells by JSRV Env protein has not been firmly determined. This issue is crucial in light of a recent study showing that mTOR can also function in the upstream of Akt and directly phosphorylates Akt at the Ser473 site in Drosophila and mammalian cells (Sarbassov et al., 2005). In this sense, it might be important to further examine the relationship between PI3K/Akt and mTOR pathways in the JSRV Env-mediated cell transformation, in particular to investigate how Akt is activated in transformed cells. On the other hand, the Akt downstream effectors have not been characterized. Although these molecules are unlikely to be directly or specifically associated with JSRV Env protein, elucidation of the downstream pathways should promote our understanding of the molecular basis of JSRV Env transformation and may provide new insights into the role of Akt pathway in cell survival and transformation.
The importance of MAPK signaling pathways for cell proliferation, differentiation, survival, and oncogenic transformation, as well as for programmed cell death or apoptosis, is becoming increasingly clear (see a comprehensive review by Pearson et al. (2001)). To date, five members of MAPK family have been identified in mammals, each playing distinct yet critical roles in a variety of cellular processes. The first is extracellular signal-regulated kinases (ERKs), the classical members of MAPK family that are activated by the upstream kinases, MAPKKKs (A-Raf, B-Raf, and Raf-1) and MAPKKs (MEK1 and MEK2). This pathway is usually activated by extracellular mitogens, and is thus important for cell proliferation and differentiation (reviewed by Ballif and Blenis (2001)). The second and third families are c-Jun amino-terminal kinases (JNKs) (JNKs 1, 2 and 3) and p38 (isoforms α, β, γ and δ), respectively. These two pathways are often activated by environmental stress, UV radiation and cytokines, and therefore are critical for normal immune and inflammatory responses (reviewed by Wada and Penninger (2004)). The other two members are ERKs 3/4 and ERK5, respectively, with biological function having not been well characterized. Notably, ERK5, also called big MAPK or BMK1 because of its large size, has been shown to be important for neuronal survival (reviewed by Cavanaugh (2004)). Among these MAPK family members, the Raf-MEK-REK1/2 pathway has been shown to play the most critical roles in cell transformation by oncogenes, including oncogenic retroviruses.
The roles of the MAPK pathway in JSRV Env transformation has just begun to be appreciated. We initially examined ERK1/2 phosphorylation in 208F cells transformed by JSRV Env protein, but failed to detect any ERK phosphorylation in these cells (Liu et al., 2003b). A similar observation was also recently made by Fan and colleagues in the Env-transformed NIH 3T3 cells (Maeda et al., 2005). Interestingly, the latter group found that the MEK1-specific inhibitor, PD98059, as well as an H/N-Ras inhibitor, FTI-277, can inhibit Env transformation of NIH 3T3 and RK3E rat epithelial cells in a dose-dependent manner (Maeda et al., 2005), indicating that the Ras-Raf-MEK-ERK pathway might be involved in JSRV Env transformation of these cells. However, it is still not known how this pathway is activated by the JSRV Env protein and why ERK phosphorylation is not detected in Env-transformed cells. One possibility is that ERK phosphorylation is transient or unstable in the transformed cells, either due to the presence of negative feedback loops or cross-talks with other signaling pathways (see details below).
MAPK/p38 pathway was also recently shown to be involved in JSRV Env-mediated cell transformation. This is directly demonstrated by the effect of p38 inhibitor SB203580 that can increase the Env transformation efficiency in NIH 3T3 and RK3E cells (Maeda et al., 2005). Interestingly, p38 phosphorylation is only slightly increased in the Env-transformed cells compared to that of negative controls (Maeda et al., 2005), which suggests additional pathways that may regulate p38 phosphorylation. Intriguingly, treatment of transformed NIH 3T3 cells with SB203580 was shown to significantly potentiate MEK1/2 and ERK1/2 phosphorylation in these cells (Maeda et al., 2005), implying a cross-talk between ERK and p38 pathways. While this may explain why ERK phosphorylation is not detectable in the Env-transformed NIH 3T3 and 208F cells (Liu et al., 2003b; Maeda et al., 2005), exactly how p38 pathway negatively modulates ERK phosphorylation remains to be better defined. One possibility is that p38 activates PP1/2A, a serine/threonine phosphatase that dephosphorylates MEK1/2, leading to reduced ERK1/2 phosphorylation (Westermarck et al., 2001). Alternatively, ERK1/2 may be dephosphorylated by other unknown phosphatases. Additional question that needs to be seriously addressed is how JSRV Env protein activates the p38 pathway in NIH 3T3 and RK3E cells, and whether or not this pathway is important for Env transformation in other cell lines. It is noteworthy that although ERK/MAPK phosphorylation is readily present in the natural and experimentally-induced OPA, however, p38 phosphorylation is not detected in these tumors (Maeda et al., 2005).
The mechanism of JSRV Env transformation in the immortalized human bronchial epithelial cell line BEAS-2B is quite different from that in fibroblasts and other epithelial cells. Several lines of evidence indicate that the receptor tyrosine kinase RON and the cell-entry receptor Hyal2 are important for BEAS-2B cell transformation by the JSRV Env protein (Danilkovitch-Miagkova et al., 2003). In normal BEAS-2B cells, Hyal2 is constitutively associated with RON and inhibits RON activation. Expression of JSRV Env leads to Env binding to Hyal2, Hyal2 degradation, liberation of RON from Hyal2 inhibition, and RON activation and cell transformation (Danilkovitch-Miagkova et al., 2003). In this case, Hyal2 functions as a tumor suppressor that negatively regulates RON activity and Env-mediated cell transformation. Importantly, a dominant-negative kinase-dead RON mutant was able to block transformation by Env, indicating that the RON-Hyal2 pathway was critically important for Env transformation of BEAS-2B cells.
RON belongs to the Met proto-oncogene family, and is widely expressed in human tissues, in particular those of epithelial origin and in immune cells (reviewed by Comoglio and Boccaccio (1996)). RON is also overexpressed in a variety of human tumors, in particular breast and colon cancers (reviewed by Wang et al. (2003)). Overexpression of RON has been shown to induce distal lung tumors in transgenic mice and synergize in mammary tumor formation and metastasis (Wang et al., 2003). The role of RON in BEAS-2B transformation by JSRV Env is supported by RON tyrosine phosphorylation in transformed cells and by the fact that the dominant-negative kinase-dead RON mutant could block cell transformation by JSRV Env (Danilkovitch-Miagkova et al., 2003).
Several critical questions need to be addressed concerning the RON-Hyal2 pathway. The first is whether a constitutively activated RON can directly transform BEAS-2B cells in the absence of JSRV Env. We have attempted to address this issue by overexpressing RON in BEAS-2B cells, but failed to observe morphological transformation. We also expressed RON in several cell lines, including NIH 3T3, 208F and MDCK, again, we only detected low transforming activity (Miller et al., 2004; unpublished results). These results indicate that human RON has low if any transforming activity, and are consistent with a previously published study (Santoro et al., 1996). However, constitutively activated RON mutants have been shown to induce transformation in vitro, cause tumors in nude mice, and promote tumor metastasis (Wang et al., 2003).
The involvement of Hyal2 in JSRV Env transformation of BEAS-2B cells is supported by the disassociation of the Hyal2-RON complex that is normally detected in the untransformed cells (Danilkovitch-Miagkova et al., 2003). This finding is intriguing, because it indicates that Hyal2 can function as a tumor suppressor through RON. We have further investigated this issue by overexpressing human Hyal2 in NIH 3T3, 208F or MDCK cells that express human RON (note that these cells do not normally express an ortholog of human RON), but unfortunately we found no obvious effect of Hyal2 on the RON-induced scattering and tyrosine phosphorylation (Miller et al., 2004; unpublished results). We also overexpressed human Hyal2 in several human lung cancer cell lines by retroviral vector transduction, and again, no significant change in cell morphology and proliferation was observed (unpublished results). Together, these experiments failed to support for the role of Hyal2 in inhibiting RON activity, in agreement with a previous study showing that Hyal2 was unable to suppress the growth of normal human bronchial epithelial cells and lung cancer cell lines (Ji et al., 2002). Interestingly, the latter group did observe a 70% reduction of lung tumor metastasis following expression of Hyal2 in an experimental A549 metastatic lung cancer model (Ji et al., 2002). Thus, whether or not Hyal2 is a general tumor suppressor cannot be easily answered at present.
While apparently essential for BEAS-2B cell transformation by JSRV Env, the RON-Hyal2 pathway is unlikely to operate in NIH 3T3 cells. First, the mouse ortholog of RON, Stk, is not expressed in NIH 3T3 cells, as determined by showing that radioactively-labeled MSP, the RON/Stk ligand, does not bind to NIH 3T3 cells, and by showing that Stk protein cannot be detected in lysates from NIH 3T3 cells by Western blotting analysis (Wang et al., 1995; Miller et al., 2004). Second, mouse Hyal2, although expressed in NIH 3T3 cells, does not appear to bind JSRV Env protein, as shown by FACS assay using a hybrid protein consisting of JSRV SU fused to a human IgG Fc fragment (Liu et al., 2003a). Third, overexpression of mouse Hyal2 has no effect on JSRV Env transformation efficiency of NIH 3T3 cells, nor has any effect on the transformed phenotype (Liu et al., 2003a). Likewise, the RON-Hyal2 pathway is unlikely to play a role in Env transformation of MDCK cells because RON expression is not detected in MDCK cells (Wang et al., 1994), and overexpression of human RON in MDCK cells has no effect on transformation by JSRV Env (Liu and Miller, 2005). In fact, we have recently shown that JSRV Env protein transforms MDCK cells by the same mechanism as that in rodent fibroblasts (Liu and Miller, 2005). Thus the RON-Hyal2 pathway appears to be specific for JSRV Env transformation of BEAS-2B cells.
Interestingly, the RON-Hyal2 pathway might be functionally operative in some human lung cancer cell lines. This was suggested by the finding that RON is overexpressed and constitutively activated in some human lung cancer cell lines, in particular BAC cells derived from BAC patients (Danilkovitch-Miagkova et al., 2003). In addition, the receptor activity of human Hyal2 is inhibited or lost in these cells, as shown by reduced or undetectable titers of JSRV retroviral vectors as compared to that of normal human lung epithelial cells (unpublished results). One intriguing possibility is therefore that Hyal2 protein is degraded in these cells, due to the presence of JSRV or a JSRV-like virus or sequence. Alternatively, defects may exist in the Hyal2 genomic DNA or mRNA of these BAC cells that preclude the Hyal2 protein synthesis. Future effort should focus on addressing these possibilities, and results from these investigations should lead to a better understanding the mechanism of RON activation in these BAC cells and a possible relationship between JSRV and human lung cancer.
A mouse model of lung cancer for JSRV Env oncogenesis
The inability of JSRV Env to mediate infection of mouse cells (Rai et al., 2000, 2001) or to bind mouse Hyal2 (Liu et al., 2003a), and the apparently essential role of Hyal2 in Env transformation of BEAS-2B human lung epithelial cells (Danilkovitch-Miagkova et al., 2003) initially discouraged an effort to establish a mouse model for JSRV oncogenesis. However, growing evidence that JSRV Env protein could transform a variety of cell types, including epithelial cells, independently of Hyal2 (Liu and Miller, 2005; Maeda et al., 2005; Varela et al., 2006), suggested that development of such a mouse model might be possible. Indeed, a mouse model of lung cancer induction by JSRV Env has been recently developed by using a replication-defective adeno-associated virus type 6 (AAV6) vector that expresses the JSRV Env protein (Wootton et al., 2005). AAV6 vectors promote long-term transgene expression in all epithelial cell types of mouse lungs (Halbert et al., 2001), including type II pneumocytes, a presumed target of JSRV oncogenesis in sheep. Mouse lung tumors induced by JSRV Env appeared as early as 9 weeks after vector exposure, with size increasing over time, and showed morphology and localization similar to that in sheep (Wootton et al., 2005) (Figure 3).
AAV6 vectors are known to transduce all types of epithelial cells in the mouse lung and airway. Strikingly, tumors induced by JSRV Env-expressing AAV6 vector all expressed the type II pneumocyte marker, surfactant protein C (Wootton et al., 2005). It has been previously reported that the JSRV long-terminal repeat (LTR) promoter and enhancer is preferentially active in the type II pneumocytes (Palmarini et al., 2000a; McGee-Estrada et al., 2005), due to a requirement for the presence of hepatocyte nuclear factor-3 beta (HNF-3β) and other factors expressed in type II cells (McGee-Estrada et al., 2002) that can transactivate the JSRV LTR and enhance the downstream gene expression (McGee-Estrada and Fan, 2006). While this proposed mechanism may explain the tumor type observed in sheep, it cannot explain the tumor type observed in the mouse model because in this case, expression of JSRV Env protein is not driven by the native JSRV LTR promoter but by the broadly-active Rous sarcoma virus promoter (Wootton et al., 2005). One possibility, yet to be proved, is that the restricted JSRV Env expression in cells expressing type II pneumocyte markers in mouse lungs reflects a general feature of mouse lung biology. It has been noticed for years that the majority of murine lung tumors induced by oncogenes or chemical carcinogens contain cells having the phenotypes of type II pneumocytes or Clara cells, but the exact mechanism is not well understood (Tuveson and Jacks, 1999).
Another important finding from the mouse model for JSRV Env oncogenesis is that host immunity can provide a significant level of protection against tumorigenesis. This is manifested by the fact that AAV6 vector expressing JSRV Env induced multiple large lung tumors in the immune-deficient C57BL/6 Rag2 mice, but yielded a few small tumors in immune-competent C57BL/6 mice (Wootton et al., 2005). In addition, serum harvested from the C57BL/6 mice exposed to the AAV6 vector encoding JSRV Env is able to block transduction by a JSRV retroviral vector (Wootton et al., 2005), indicating that a host humoral immune response was indeed mounted against JSRV Env. In contrast, normal sheep are immunotolerant of JSRV and are susceptible to JSRV oncogenesis (DeMartini et al., 2003) because of the presence and expression of several closely related endogenous retroviruses in their genomes (Hecht et al., 1996; Palmarini et al., 2000b; Sanna et al., 2002; Klymiuk et al., 2003; Spencer et al., 2003). Indeed, no humoral or cellular immune response has been detected against JSRV, either in the naturally occurring or experimentally induced sheep (Sharp and Herring, 1983; Palmarini et al., 1996; Ortin et al., 1998; Summers et al., 2002). Thus, it is possible that host immune response can protect other species, including humans, from JSRV tumorigenesis.
Several important questions remain to be answered with respect to the mouse model of JSRV Env oncogenesis. First, and as mentioned above, it is still unknown why tumor formation is restricted to peripheral cells that express type II pneumocyte markers, and it will be important to determine whether this is JSRV-Env-specific or a reflection of the default mode of mouse lung cancer. Second, the molecular mechanisms of oncogenic transformation by JSRV Env in the mouse model are not known. It will be essential to determine whether the oncogenic domains and signaling pathways identified in vitro are also active in the mouse model of JSRV Env oncogenesis. While it is possible that PI3K/Akt and Ras-Raf-MEK-MAPK pathways are still important for JSRV Env tumorigenesis in vivo, other signaling pathways may also play a role and therefore should be explored. On the other hand, although mouse Hyal2 does not interact with JSRV Env, making a role for the RON-Hyal2 pathway unlikely, this assumption should be experimentally tested. Third, it is well accepted that tumorigenesis in vivo is a complicated process involving series of signaling events, and it would be important to examine if other factors, such as cellular oncogenes or tumor suppressor genes, might be also involved in JSRV oncogenesis in mice. Lastly, it will be interesting to determine if tumor metastasis occurs in the mouse model of lung cancer induced by JSRV Env. Further exploration of the mouse model for JSRV Env oncogenesis will provide critical insights into the mechanism of sheep retroviral oncogenesis and may illuminate the biology of human lung cancer.
Concluding remarks and perspectives
Cancer is a multistep process involving complex interactions between activation of oncogenes, inactivation of tumor suppressor genes, as well as genetic or epigenetic alterations associated with tumor progression and metastasis (Hanahan and Weinberg, 2000). While JSRV is an acute transforming retrovirus, with its Env protein acting as a potent oncogene, it must be born in mind that tumor development in sheep by JSRV usually takes months to years in natural settings (Sharp and DeMartini, 2003). This paradox highlights, once again, the multistep nature of tumorigenesis by JSRV in sheep; more importantly, it suggests other mechanisms, such as insertional mutagenesis, might also play a role in JSRV oncogenesis. Several groups have investigated the possibility that JSRV provirus may have common integration sites in the sheep genomes, and obtained interesting but inconsistent results (DeMartini et al., 2001; Cousens et al., 2004; Philbey et al., 2006). Notably, one common integration site was recently identified on the sheep chromosome 16 (Cousens et al., 2004), and another clonal integration was observed in the receptor protein tyrosine phosphatase γ genes of chromosome 19 (Philbey et al., 2006). However, the significance of these integration sites for JSRV oncogenesis remains to be further defined.
As discussed in this review, significant progress has been made over the last few years in understanding JSRV oncogenesis. However, our understanding in the mechanisms of cell transformation by JSRV Env protein is still far from complete. For instance, we still do not know how the JSRV Env protein engages the cell signaling network, leading to transformation, and identification of cellular molecules that directly interact with JSRV Env protein will be of great interest. On the signaling side, three major pathways have been uncovered, but how they are activated independently yet cross-talk to each others is not known. In addition, we know little about JSRV Env transformation in primary lung epithelial cells and in vivo, and future research should be directed towards this end.
JSRV presents an attractive model for the study of human lung cancer, given the strong similarities between the sheep tumors induced by JSRV and human adenocarcinoma. The convergence and interplay among JSRV Env, human Hyal2, and RON, as revealed in the BEAS-2B cell transformation by JSRV Env (Danilkovitch-Miagkova et al., 2003), are very interesting and may have important implications in this respect. While traditional methods such as PCR or immunohistochemistry may be valuable, future efforts should take advantage of other recently developed technologies, such as microarray or proteomics, to explore whether JSRV, or a related virus, is involved in human lung cancer. Discovery of such a virus, although challenging, would have a profound impact on lung cancer prevention, diagnosis and treatment. Even if no such virus is found, these investigations should lead to a deeper understanding of retroviral oncogenesis as well as human lung tumorigenesis.
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We apologize to those colleagues whose original work is not cited due to space limitations. We thank Michael Lerman, Alla Danilkovitch-Miagkova and James DeMartini for collaborations and discussions. This work was supported by funds from the Canadian Institutes of Health Research and McGill University to S-L Liu, and grants from the United States National Institutes of Health and the Fred Hutchinson Cancer Research Center to AD Miller, S-L Liu is a Canada Research Chair in Virology and Gene Therapy.
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Liu, SL., Miller, A. Oncogenic transformation by the jaagsiekte sheep retrovirus envelope protein. Oncogene 26, 789–801 (2007). https://doi.org/10.1038/sj.onc.1209850
- Jaagsiekte sheep retrovirus
- envelope protein
- lung cancer
- signaling pathways
- mouse model
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