The studies presented here demonstrate a system to transfer cDNAs into premalignant lesions in mice to analyze their effects on tumor progression. This system utilizes RCASBP avian retroviral vectors to deliver cDNAs into pancreatic β cells in RIP-Tag; RIP-tva bitransgenic mice during tumorigenesis. This approach has the advantage of introducing somatic genetic changes in a tissue-specific and time-controlled fashion, which more faithfully mimics sporadic tumor development. It also avoids any potential perturbation of normal tissue formation due to the ectopic expression of the gene of interest during development. It is much faster to generate vectors carrying genes of interest than to generate transgenic mice. In addition, using cell lines derived from β-cell tumors in RIP-Tag; RIP-tva bitransgenic mice, further biochemical and phenotypic analysis can be performed, taking advantage of the high efficiency of viral infection in vitro.
The low efficiency of in vivo infection has been a limitation of using the RCAS-TVA system [13
]. Here, we were able to achieve 10%–20% in vivo infection efficiency in hyperplastic pancreatic islet β-cell lesions using high viral titer (>108
infectious units per milliliter) and the intra-cardiac route to deliver the RCASBP viruses into mice. Since not all the premalignant lesions are infected with the RCASBP vectors and 2%–4% of islets develop into tumors in the RIP-Tag; RIP-tva
bitransgenic mice without viral infection, only the factors that confer a selective advantage over the natural course of tumorigenesis in RIP-Tag; RIP-tva
mice can be identified. Moreover, the percentage of RCASBP-Bcl-xL
-positive tumors decreases over time as the uninfected RIP-Tag; RIP-tva
cells gradually develop into tumors. In particular, RCASBP vectors carrying genes that promote metastasis will be most easily recognized, because metastasis to pancreatic lymph nodes or other organs does not normally occur in RIP-Tag; RIP-tva
mice. Although tumors are likely to be clonally expanded from single cells infected with RCASBP vectors carrying progression factors, we were not able to show whether the tumors were clonal because of the limited DNA materials isolated from the small tumors in this study.
We delivered RCASBP viruses carrying tumor progression factors into RIP-Tag; RIP-tva
bitransgenic animals at 7 wk of age, when hyperplastic islets develop. Analogous to the results obtained from RIP1-Tag2; RIP1-dnE-cad
bitransgenic mice [22
], we demonstrated that introduction of RCASBP-dnE-cad
into the hyperplastic islets of RIP-Tag; RIP-tva
animals promoted invasive tumor formation and metastases to the pancreatic lymph nodes. Mice at other ages could also be used to study whether the time at which the candidate factors are introduced influences the outcome, as long as islet cells undergo cell division to allow infection. For example, the transforming growth factor β1 (TGFB1) receptor can act as a tumor suppressor at early stages of tumor development, but at later stages TGFB1 responsiveness promotes invasion and metastasis [41
]. Moreover, a combination of several RCASBP viruses encoding different factors of interest could be used for infection simultaneously or sequentially, and the proviral DNA could be isolated from especially metastatic malignant tumors to identify single factors or combinations of factors that may contribute to the malignancy. However, this approach is limited by the inability of the RCASBP vector to accommodate cDNAs greater than 2.5 kb in size.
The effects of overproducing Bcl-xL via somatic gene transfer were provocative and unexpected. First, no significant protection against apoptosis was found either in the tumors from mice infected with RCASBP-Bcl-xL
or in the tumor cells infected with RCASBP-Bcl-xL
in vitro. These findings are quite distinct from the demonstrable anti-apoptotic effect seen when Bcl-xL was expressed via a transgene in all islet β cells throughout tumorigenic ontogeny in the RIP1-Tag2
], as well as in MYC transgenic mice [26
]. It remains possible that Bcl-xL contributes to cell survival at earlier stages (hyperplasia/angiogenesis) or a later stage (metastasis) in mice infected with RCASBP-Bcl-xL
. Additionally, we cannot exclude episodic effects on apoptosis when tumors were forming, as opposed to chronic effects detectable at the later time points of our analysis. This apparent insensitivity of β-cell tumors to the anti-apoptotic effect of Bcl-xL deserves future investigation. Notably, a similar phenotype of increased invasiveness without suppression of apoptosis has been observed when another apoptotic modulator was overexpressed in the RIP1-Tag2
mice. Exogenous production of IGF1R for the IGF1/2 survival factors in all islet β cells caused increased invasiveness and lymph node metastasis, with increased (not decreased) apoptosis in the premalignant stages, while having no impact on the tumor stages [23
]. Yet elimination of the gene encoding IGF2 produced highly apoptotic tumors with reduced growth, demonstrating the importance of anti-apoptotic signaling for islet tumorigenesis [43
]. The similar consequences of ectopically expressing Bcl-xL and IGF1R may imply mechanistic similarity that should be further explored.
Second, we observed that the actin cytoskeleton was rearranged in invasive tumors infected with RCASBP-Bcl-xL, presumably contributing to increased motility. In addition, tumor cells infected with RCASBP-Bcl-xL in vitro displayed altered morphology, abnormal cortical distribution of the actin cytoskeleton, and elongated cell shape. The morphological changes in tumor cells infected with RCASBP-Bcl-xL may contribute to enhanced cell migration and invasion as observed in the two-chamber assays.
Third, as measured by Western blotting and cDNA microarray expression profiling, the epithelial cell–cell junction protein, cadherin 1, was down-regulated and a transcriptional repressor of cadherin 1, snail homolog 1, was up-regulated in the tumor cells infected with RCASBP-Bcl-xL. These changes may contribute to the rearrangement of the actin cytoskeleton and diminish the strength of cellular adhesion. Indeed, tumor cells infected with RCASBP-Bcl-xL exhibited a strong capability to migrate through transwell membrane and to invade through Matrigel, but the mechanism by which Bcl-xL regulates cadherin 1 remains to be determined.
Finally, the identification of a novel Bcl-xL-interacting protein, myosin Va, may help to explain the pro-invasive activity of Bcl-xL. Myosins belong to a superfamily of actin-based motor proteins comprising at least 15 classes [39
]. There are two main groups of myosins: the conventional, well-characterized myosin II class of muscle and non-muscle cells and the unconventional myosins. The myosin II class is referred to as “conventional myosin” because for many years this was the only class of myosin known; members of this class are able to form filaments and are involved in muscle contraction, cell migration, and cytokinesis. The functions of most of the unconventional myosins are not known, but some have been shown to participate in the extension of processes at the leading edge of crawling cells. For example, myosin I is required for formation of pseudopod extensions in the amoeba Dictyostelium,
and myosin V for extension of filopodia in neurons [44
By comparing the effects of suppressing the function of cadherin 1 with dnE-cad and overproducing Bcl-xL, we have highlighted the provocative pro-invasive activity of Bcl-xL. Cadherin 1 is a well-established barrier to invasive growth in many other epithelial cancers [46
], and its suppression enables invasive growth of pancreatic β-cell tumors in transgenic mice [22
]. Yet Bcl-xL produced a more highly invasive phenotype in a Matrigel invasion assay of cultured tumor cells than did dnE-cad, supporting the proposition that Bcl-xL has roles other than suppression of cadherin 1 that enhance the invasive growth phenotype. The levels of endogenous cadherin 1 in these two types of cells could contribute to the differences. Whereas endogenous cadherin 1 protein levels were not affected when the dominant-negative form was introduced into the cells, cadherin 1 was down-regulated in the tumor cells infected with RCASBP-Bcl-xL
. This effect could reduce cell–cell adhesion and also affect the organization of the actin cytoskeleton. On the other hand, the frequency of lymph node metastasis was similar in RIP-Tag; RIP-tva
mice infected with RCASBP-Bcl-xL
. It is possible that the β-cell tumor microenvironment in vivo is less discriminating than Matrigel, a solubulized basement membrane preparation extracted from a mouse sarcoma [47
The BCL2 family proteins function in carcinogenesis by preventing apoptosis of tumor cells, instead of promoting cell proliferation [48
]. Of the BCL2 family members, BCL2 and Bcl-xL are most closely related to each other, and repress cell death through common mechanisms [49
]. However, several lines of evidence indicate that they are not functionally equivalent in tumorigenesis. In primary breast cancer, overexpression of Bcl-xL
is associated with higher tumor grade and nodal metastasis, and overexpression of Bcl2
is correlated with lower tumor grade and smaller tumor size [51
]. Moreover, nude mice with orthotopic implants of human breast cancer cells transfected with Bcl-xL, but not BCL2, develop lymph node metastasis [52
]. Our findings suggest that Bcl-xL enhances cell motility, remodels the actin cytoskeleton, down-regulates cadherin 1, and interacts with myosin Va without affecting cell proliferation or apoptosis. It remains to be investigated whether these properties are unique to Bcl-xL among the BCL2 family members.
In conclusion, we have developed a mouse model to assess the effect of candidate genes in tumor progression, without the need to generate conditional transgenic lines in which genes of interest are expressed at different stages of tumorigenesis. Thus far, we have employed this approach to improve our knowledge of the contribution of Bcl-xL to tumor invasion and metastasis. We postulate that Bcl-xL has a pro-invasive function other than its anti-apoptotic activity. Given that many somatic gene mutations and altered expression of thousands of genes have been discovered in cancers, it will not be a simple task to verify that each candidate gene is important during carcinogenesis. By delivering libraries of retroviruses encoding candidate factors or inhibitory RNAs into transgenic mice, it may be possible to screen many of the genes for effects during tumorigenesis in a variety of tissues.