Rat mammary carcinogenesis induced by in situ expression of constitutive Raf kinase activity is prevented by tethering Raf to the plasma membrane

Daniel R. McFarlin and Michael N. Gould1

McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706, USA

1 To whom requests for reprints should be addressed Email: gould{at}oncology.wisc.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Mammary carcinogenesis induced through expression of activated Raf was investigated using a model in which retroviral vectors were infused into the central ducts of rat mammary glands. This model allows efficient expression of experimental proteins in a small fraction of endogenous mammary epithelial cells in situ. We previously reported that Raf is the dominant oncogenic signaling pathway from activated Ras in rat mammary glands. We show here that mammary gland carcinogenesis is rapidly induced by the expression of c-Raf-1 kinase that is activated by N-terminal truncation ({Delta}-Raf). Interestingly, targeting Raf to the plasma membrane via C-terminal fusion with Ras membrane localization signals (Raf-Caax) induces Raf kinase activity that transforms 3T3 cells more frequently than {Delta}-Raf, yet in situ expression of Raf-Caax does not induce mammary carcinomas. To investigate these contrasting results and begin elucidating the mechanisms of Raf-induced mammary carcinogenesis, we combined both activating mutations (N-terminal truncation and C-terminal membrane localization motifs) in one Raf construct ({Delta}-Raf-Caax). While {Delta}-Raf-Caax transforms 3T3 cells more efficiently than {Delta}-Raf or Raf-Caax, in situ expression of {Delta}-Raf-Caax does not induce carcinomas in vivo, demonstrating that lipid modification on the C-terminus of {Delta}-Raf negates its oncogenic potential in rat mammary gland.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Members of the Ras family of genes are some of the most frequently activated and well-characterized oncogenes occurring in human cancer (1). Frequent detection of Ras mutation in human cancer and in rodent models has inspired characterization of the effector pathways through which Ras activation induces carcinogenesis. Mutational activation of Ras is well documented in rat mammary gland carcinogenesis (24) and our work expressing Ras effector loop mutants in situ suggests that Raf is the dominant oncogenic signaling pathway from activated Ras in rat mammary glands (5). E38-V12-Ras is an activated Ras effector loop mutant that lacks affinity with many Ras effector proteins (e.g. PI3K and RalGDS) but retains affinity with Raf (6). This restricted mutant induces mammary carcinomas in the rat at 55% the frequency of constitutively active V12-Ras (5). Since E38-V12-Ras carcinoma induction could possibly result from other postulated or yet to be elucidated Ras effector pathways acting synergistically with Raf, it is important to directly test Raf itself in this mammary gland carcinogenesis model.

The oncogenic potential of Raf activation had already been characterized long before it was known to associate with Ras. The Raf kinase family was first discovered as a transforming gene in murine sarcoma virus 3611(v-Raf) (7). The N-terminal half of the v-Raf protein is homologous with retroviral gag proteins and is myristoylated on the N-terminus. This results in membrane localization of v-Raf. The C-terminal half of v-Raf has high homology with the three Raf family members expressed in humans (c-Raf-1, A-Raf and B-Raf), which are generally in the cytoplasm (8). Expression of A-Raf and B-Raf is limited, while c-Raf-1 is ubiquitous (8). The N-terminal regions of cellular Raf proteins contain regulatory elements not present in v-Raf. Membrane localization and lack of N-terminal regulatory elements are both believed to contribute to the oncogenicity of v-Raf. Constitutive c-Raf-1 kinase activity and 3T3 transformation are stimulated by either N-terminal truncation ({Delta}-Raf) (9) or C-terminal fusion to Ras membrane localization signals (Raf-Caax) (10,11). While 3T3 fibroblasts provide a convenient model for rapid characterization of transforming genes, they are not able to fully model the highly ordered three-dimensional structure of polarized epithelial cells in vivo. Delivery of constitutively active Raf kinase constructs into endogenous epithelial cells in situ using replication-defective retroviral vectors infused into the central mammary ducts of the rat provides a good in vivo model (12) for characterization of Raf in carcinogenesis. Here, we use this retroviral infusion model to express several forms of constitutively active Raf kinase in vivo, to determine if Raf activation is sufficient to induce mammary carcinogenesis. Our data suggest that for Raf to induce carcinogenesis in vivo, more rigorous requirements must be met than those necessary for neoplastic transformation in cultured cells.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Vector construction
tag-Raf-K-Caax cDNA, kindly provided by C.J.Marshall, was subcloned from pmt-SM-myc-Raf-CAAX into vector pJR (with an intermediate subcloning into pBluescript II SK+ between NotI and EcoRI sites) to generate pJR-tag-Raf-K-Caax. The pJR-tag-Raf-K-Caax vector was generated as follows. With pJR-H-Ras as a template, PCR was used to amplify the 3'-terminal 60 bp of the H-Ras coding sequence (H-Caax) with useful restriction sites. One primer (5'-aaaacccgaggctgcctgtcttcaaactgaacccgcctgatg-3') has Raf codons from the AvaI site to its last amino acid codon, transitioning in-frame to H-Ras codons 170–175. The other primer (5'-attactcgagtcaggacagcacacacttg-3') generates a XhoI site, and complements the H-Ras stop codon and the last 5 amino acid codons (185–189). The resulting H-Caax PCR product was ligated into pBSK-tag-Raf-K-Caax with the K-Caax removed, resulting in pBSK-tag-Raf-H-Caax. Next, tag-Raf-H-Caax cDNA was subcloned into vector pJR to produce pJR-tag-Raf-H-Caax (Figure 1). pJR-tag-Raf was similarly constructed, except oligonucleotides 5'-aaaacccgaggctgcctgtcttctgaattcctcgagaaaa-3' and 5'-ttttctcgaggaattcagaagacaggcagcctcgggtttt-3' were annealed to reproduce the 3'-end of wt-Raf cDNA from the AvaI site to the stop codon, with EcoRI and XhoI sites added for identification and subcloning. The tag on the N-terminus of these three constructs is a 10 amino acid epitope tag from Myc that was included on the originally published Raf-CAAX (renamed tag-Raf-K-Caax in Figure 1 and throughout for clarity) for immunohistochemical visualization and protein isolation.



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Fig. 1. Linear representations of expressed Raf constructs, and their respective abilities to neoplastically transform 3T3 cells in culture (Figure 4) and mammary epithelial cells in situ. The location of the Ras binding domain (RBD), the cysteine rich region (C rich), the phosphatidic acid binding region (PAbr) and several phosphoregulating amino acids are indicated. The tag on the N-terminus of some constructs is a 10 amino acid epitope tag from myc, which was included in the originally published Raf-CAAX (9). {Delta}-Raf vectors lack amino acids 2 through 307 of c-Raf-1. Raf-K-Caax and Raf-H-Caax constructs have the last 20 amino acids of K-Ras or H-Ras respectively, fused to their C-termini, signaling for farnesylation of Raf-K-Caax proteins and farnesylation and palmitoylation of Raf-H-Caax proteins. All constructs were expressed from pJR replication-defective retroviral vectors.

 


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Fig. 4. Highly contact inhibited Swiss 3T3 cells infected with retroviral vectors expressing various constitutively active Ras or Raf constructs and the resulting transformation frequencies respectively. By 12 days post infection, individual cells expressing E12-H-Ras (A) or V12-H-Ras (B) have generated dense cell mats or clusters of foci. 3T3 cells infected with {Delta}-Raf-H-Caax (C) or with {Delta}-Raf-K-Caax (D) also form foci clusters but the individual foci within the cluster seem smaller than those induced by activated Ras. Raf-K-Caax (E) and Raf-H-Caax (F) induce foci clusters and dense cellular mats about half as often as membrane targeted Rafs lacking N-terminal regulatory regions ({Delta}-Raf-H-Caax and {Delta}-Raf-K-Caax, panel M). E38-V12-Ras infected 3T3 cells (G) also form foci clusters at a fairly high frequency. Foci induced by {Delta}-Raf expression (H) did not generally subdivide into foci clusters. Cells infected with tag-Raf-K-Caax (I) also form foci clusters, while cells infected with tag-Raf-H-Caax (J), tag-Raf (K) or wild type Raf (L) generally stop dividing after reaching confluence. The frequency of transformation (M) is the mean number of foci with stacking nuclei (from at least two trials), divided by the mean number of G418 resistant colonies induced by that vector (from at least three trials).

 
A 5' primer that generates a NotI site and Kozac consensus sequence, with the first five codons of Raf (5'-aaaagcggccgccaccatggagcacatacagggagc-3') and a 3' primer complementing the last few codons of H-Ras (5'-attactcgagtcaggacagcacacacttg-3', as used above) were used with pBSK-tag-Raf-H-Caax as a template to generate Raf-H-Caax without a tag. Raf-K-Caax without a tag was generated using pBSK-tag-Raf-K-Caax as template, the same 5' primer and a 3' primer that complements pBSK from base pair 677 through the EcoRI site, but changes the ClaI site to XhoI (5'-gacggtctcgagaagcttgatatcgaattc-3'). Raf without a tag was generated using the same 5' primer and a 3' primer that complements the last six codons of Raf (5'-ttttctcgaggaatctagaagacaggcagcctcggg-3'). A different 5' primer that also generates a NotI site and Kozac consensus sequence, but with codon 1 transitioning in-frame to codons 308–312 of Raf (5'-aaaagcggccgccaccatgccgaaaacccccgtgcc-3') was used with the same corresponding 3' primers to generate {Delta}-Raf-H-Caax, {Delta}-Raf-K-Caax and {Delta}-Raf. All six PCR products were subcloned directly into pJR (Figure 1). The construction of pJR-E12-H-Ras (13), pJR-V12-H-Ras (5) and pJR-E38-V12-H-Ras (5) have been described previously.

Vector preparation and mammary infusion
Retroviral vector preparation, culture conditions, titers, helper virus test, viral concentration, mammary gland infusions, weekly gland palpations and animal follow-up were all performed by previously described methods (5,12). Briefly, Psi-Cre packaging cells were transfected with JR retroviral plasmid constructs. The resulting ecotropic packaged JR retroviral vectors were used to infect PA317 cells for production of amphotropic retroviral vectors suitable for infection of in situ rat mammary epithelial cells. Helper virus-free retroviral concentrates were diluted to equal titers (~1 x 107 c.f.u./ml) for infusion. The central duct of each mammary gland of 50–60 day old virgin female Wistar-Furth (Harlan Sprague–Dawley) rats was infused with 15 µl of retroviral suspension. Rats were palpated weekly with location and diameter estimates of all palpable mammary tumors >3 mm being recorded. Weekly palpations were generally performed for at least 16 weeks following mammary gland infusions; a limited number of Raf-Caax rats were palpated for more than a year. At the end of each experiment, all mammary glands were dissected and visually inspected for tumors, hyperplasia or other abnormalities. Tumors/hyperplasia as small as 1 mm in diameter would be revealed by visual inspection of a dissected gland. For each vector that failed to produce palpable tumors, several glands (between 4 and 8) from separate rats were sectioned. Glands were dissected, spread out for fixation in formalin, embedded in paraffin, sectioned flat and hematoxylin and eosin stained for microscopic inspection. The effect of virus type on the number of positive glands per animal was assessed using a generalized linear model assuming Poisson variability. The log-rank test was used to test for an effect of virus type on the time to first tumor.

3T3 transformation assay
Highly contact-inhibited Swiss 3T3 cells were maintained at very low density (split 1:40 and 1:80 when cell processes start to make contact). At the time of cell–cell contact, 3T3 cells from multiple plates were mixed (for uniformity) to prepare plates of cells for infection. Two days later, just prior to infection, cells were harvested from several plates to determine the representative number of cells per plate. Previously titered retroviral concentrates were thawed and diluted to equal titers, then serially diluted to 5 x 105 and 2.5 x 104 G418r c.f.u./ml retroviral stocks to use for infection. Diluted stocks were added with 5 µg/ml polybrene to infect 3T3 cells. Twelve days later each plate was scored for the number of cell colonies with stacked nuclei. Colonies with stacked nuclei are visible (without magnification) as white papilloma-like projections, while areas of cellular crowding (loss of contact inhibition) are refractive to light. To score the number of transformations per plate, refractive areas were marked then phase contrast light microscopy was used to distinguish nuclear stacking from cellular crowding.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Activated Raf, in the form of {Delta}-Raf, was found to induce mammary carcinogenesis in vivo. The latency for {Delta}-Raf tumor induction was not significantly different from that of H-Ras activated by mutation of codon 12 to glutamic acid (E12-H-Ras) (P = 0.898) (Figure 2A). The frequency of carcinoma induction from {Delta}-Raf expression was slightly different (P = 0.046) from E12-H-Ras (Figure 2B). Neither the latency nor frequency of {Delta}-Raf carcinoma induction was significantly different from those we have reported previously from E38-V12-H-Ras (5). In addition to valine at residue 12 (for constitutive signaling), E38-V12-Ras also has glutamic acid (rather than aspartic acid) at residue 38, causing Raf-prejudiced signaling. Histopathological evaluation revealed that the tumors induced by {Delta}-Raf were generally papillary carcinomas with characteristics similar to those induced by E12-H-Ras and other Ras constructs, including E38-V12-H-Ras (Figure 3).



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Fig. 2. Latency and frequency of mammary carcinoma induction by in situ expression of {Delta}-Raf and E12-H-Ras. (A) Latency to palpable carcinoma induction by {Delta}-Raf is not significantly different from E12-H-Ras carcinoma induction (P = 0.898). (B) Mean frequency per rat of palpable carcinoma induction by {Delta}-Raf is slightly different from E12-H-Ras carcinoma induction (P < 0.046). For each vector, all 12 mammary glands of each rat were infused with 15 µl of a standard titer of retroviral suspension. JR-{Delta}-Raf retrovirus was diluted to 1.1 x 107 ± 0.3 x 107cfu/ml for infusion. A prediluted frozen stock of JR-E12-H-Ras retrovirus (12) was used as an internal titering standard, defined at 1 x 107 cfu/ml. JR-E12-H-Ras was infused into 19 rats, a total of 228 glands. {Delta}-Raf was infused into eight rats, a total of 96 glands. Rats were palpated weekly. Location and diameter estimates of all palpable mammary tumors larger than 3 mm were recorded. The effect of virus type on the number of positive glands per animal was assessed using a generalized linear model assuming Poisson variability. The log-rank test was used to test for an effect of virus type on the time to first tumor.

 


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Fig. 3. H&E stained paraffin sections of mammary gland and mammary carcinomas induced by Ras or Raf. In the normal gland (A), adipose cells surround mammary epithelial cells. Mammary carcinomas produced by E12-H-Ras (B), E38-V12-H-Ras (C), or {Delta}-Raf (D) share similar morphological characteristics to one another. Tissues were fixed in formalin, embedded in paraffin for sectioning then stained with H&E. Mammary gland was spread out on a slide prior to fixing and sectioned flat.

 
Unlike {Delta}-Raf, expression of Raf-Caax was not able to induce rat mammary carcinomas in situ. Several Raf-Caax vectors were evaluated. Previously published Raf-Caax contains the C-terminus of K-Ras and has an epitope tag on the N-terminus (tag-Raf-K-Caax) (11). Since H-Ras is more tumorigenic in rat mammary gland than K-Ras (13) and because Ras differences lie primarily in the C-terminus, Raf with a H-Ras C-terminus (tag-Raf-H-Caax) was also constructed and expressed. These vectors were also reconstructed and expressed without epitope tags (Raf-K-Caax and Raf-H-Caax) to reduce the likelihood of immunosurveillance in vivo. Like {Delta}-Raf, all four full-length Raf-Caax constructs were able to transform 3T3 cells infected in vitro (Figure 4E, F, I and J) at 0.03–45%. Interestingly, we found that the tag on the N-terminus of Raf dramatically attenuated transformation of 3T3 cells. Raf-K-Caax and Raf-H-Caax vectors were an order of magnitude more potent at transforming 3T3 cells than {Delta}-Raf and tag-Raf-K-Caax and multiple orders of magnitude stronger than tag-Raf-H-Caax (Figure 4 M). However, unlike {Delta}-Raf, none of these four full-length Raf-Caax variants induced a single palpable tumor by infecting mammary epithelial cells in situ, in spite of repeated trials (combined total of 56 rats, 672 tumor-negative glands). Microscopic inspection of Raf-Caax-infused glands did not reveal any hyperplasia or increased epithelial cell density, although the structure of the mammary gland would make hyperplasia difficult to distinguish in sections.

Non-quantitative RT–PCR on mRNA from infected glands indicated that mammary expression of proviral RNA remained for at least several months (data not shown). The low frequency of infected mammary epithelial cells and high background staining in the rat hindered decisive immunohistochemical labeling of individual infected cells within the mammary gland. Immunofluorescent labeling of the epitope tag included in some constructs was able to confirm production of tagged proteins in infected 3T3 cells in culture.

We found that {Delta}-Raf expression caused mammary gland carcinogenesis while several forms of Raf-Caax did not. This is in stark contrast to our in vitro results, in which non-tagged Raf-Caax constructs were an order of magnitude more potent than {Delta}-Raf. As a first step to understanding the mechanisms necessary for Raf kinase activity to induce mammary carcinogenesis, we generated constructs combining both of these activating mutations. {Delta}-Raf fused to the membrane localization signals of K-Ras or H-Ras ({Delta}-Raf-K-Caax or {Delta}-Raf-H-Caax) transformed Swiss 3T3 cells with approximately twice the frequency of Raf-K-Caax or Raf-H-Caax (Figure 4 M). Like activated E12-H-Ras and V12-H-Ras, {Delta}-Raf-H-Caax transformed Swiss 3T3 cells with ~100% penetrance, more than 30 times the potency of {Delta}-Raf. However, {Delta}-Raf-K-Caax and {Delta}-Raf-H-Caax did not induce mammary carcinogenesis (combined total of 12 rats, 144 tumor-negative glands). We conclude that lipid modification on the C-terminus of Raf, which results in Raf membrane localization (10,11), Raf kinase activation (10,11) and 3T3 cell transformation, actually blocks neoplastic transformation of mammary epithelial cells in situ by active Raf.

How is it that fusion to Ras membrane localization domains can activate Raf kinase activity but block carcinogenesis induced by activated Raf? Signals generated from constitutive membrane-bound Raf kinase activity may encourage apoptosis or differentiation of mammary epithelial cells in situ, rather than neoplastic transformation. Oncogenic targets within mammary epithelial cells could be unreachable from the plasma membrane (i.e. nuclear, perinuclear or mitochondrial targets) making internalization necessary for effective oncogenic signaling. It has been suggested that endocytosis is important for effective Raf signaling (1417). Similarly, oncogenic targets within mammary epithelial cells could be unreachable from particular microdomains of the plasma membrane. Raf-K-Caax constructs are most likely localized to the ‘disordered’ plasma membrane (18), conceivably clustered in acidic microdomains produced by electrostatic interaction between the polybasic region of K-Ras and acidic lipids in the membrane (19,20). In contrast, Raf-H-Caax constructs are likely localized to lipid rafts (18). When H-Ras is activated via binding with GTP it is redistributed from rafts to the disordered plasma membrane (21). H-Ras redistribution is thought to result from GTP-induced conformational changes (21), which Raf may not provide, so Raf-H-Caax constructs could be stranded in lipid rafts. Furthermore, removal of amino acids 166–172 from H-Ras has recently been shown to prevent GTP-induced redistribution to the disordered membrane (22). All three Raf-H-Caax constructs lack the first four of the seven amino acids from this linker domain, further increasing the likelihood that Raf-H-Caax constructs were primarily localized to rafts. When Raf kinase activity was preferentially localized to rafts (H-Caax constructs) or to the disordered plasma membrane (K-Caax constructs) there was a failure to induce rat mammary cancer. A Raf construct that could mimic GTP-induced H-Ras redistribution might be able to induce carcinomas from the membrane. However, how a construct would be designed to best recapitulate the tertiary structures of this linker region, without including the majority of the H-Ras molecule, is currently unclear.

Lipid attachment on the C-terminus of Raf could also prevent carcinoma induction by preventing proper orientation of the Raf kinase domain. The serine at amino acid 621, within the kinase domain of Raf, is necessary for kinase activity; this is located just 27 amino acids from the C-terminus of Raf and less than 45 amino acids from the Raf-Caax lipid modification. In contrast, the Ras-binding domain of Raf is more than 500 amino acids from serine 621 and the lipid modification on the N-terminus of v-Raf is more than 600 amino acids away. The close proximity of the lipid modification to the Raf kinase domain may prevent Raf-Caax and {Delta}-Raf-Caax kinase activity from accessing targets critical for mammary carcinogenesis.

Characterizing cellular mechanisms necessary for carcinogenesis can facilitate development of new anticancer drugs and therapies. To date, the development of efficacious drugs that target Ras or its signaling pathways have been limited. We feel that one factor underlying this lack of success has been the choice of models for target validation. Many of the current models are in vitro with immortalized cells and often focus on fibroblastic cell lines such as 3T3. We sought to compare Raf in the 3T3 model to an in vivo model that more accurately emulates carcinogenesis. For pragmatic reasons (i.e. the availability of a quantitative carcinogenesis assay) we chose to focus on the mammary gland. Our findings strongly support a difference in Ras/Raf signaling between these two models. Whether this difference is due to location (in vitro/in vivo), cell lineage (fibroblast/epithelial), organ/tissue-specific differences or confounding effects of cellular immortalization remains to be tested.

In summary, we found that expression of activated Raf could efficiently induce rat mammary gland carcinogenesis. This supports our findings from expression of Ras effector loop mutants that Raf is the most potent oncogenic effector of Ras. While Raf activation can initiate carcinogenesis, Raf kinase activity was not oncogenic when tethered to the membrane with C-terminal lipid modification. These results suggest that complex mechanisms regulate the potential of constitutive Raf kinase activity to induce carcinogenesis. A better understanding of the mechanisms involved in Raf oncogenic activity in vivo may provide novel targets for cancer prevention and treatment.


    Acknowledgments
 
We thank Dr Mary J.Lindstrom for statistical assistance and Dr Laurie Shepel for reviewing the manuscript. This work was supported by National Institutes of Health/National Cancer Institute grant CA77527 and by US Army Medical Material Command pre-doctoral fellowship grant DAMD17-98-1-8357.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

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Received September 18, 2002; revised March 5, 2003; accepted March 8, 2003.