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[Cancer Research 60, 1552-1556, March 15, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

Transfection of Constitutively Active Mitogen-activated Protein/Extracellular Signal-regulated Kinase Kinase Confers Tumorigenic and Metastatic Potentials to NIH3T3 Cells1

Danny R. Welch2, Toshiyuki Sakamaki, Ralph Pioquinto, Timothy O. Leonard, Steven F. Goldberg, Qiufang Hon, Raymond L. Erikson, Manuel Rieber, Mary Strasberg Rieber, Deana J. Hicks, Joseph V. Bonventre and Alessandro Alessandrini2

Jake Gittlen Cancer Research Institute, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033-2390 [D. R. W., T. S., T. O. L., S. F. G., D. J. H.]; Medical Services, Massachusetts General Hospital, Charlestown, Massachusetts [R. P., J. V. B., A. A.]; Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts [Q. H., R. L. E.]; and Instituto Venezolano Investigaciones Cientificas, Caracas, Venezuela [M. R., M. S. R.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Cellular growth and differentiation are controlled by multiple extracellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinases, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylation at Ser218 and Ser222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp218], [Asp218, Asp222], and [Glu218, Glu222], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, independent of ERK activity. The transformed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp218] or [Asp218, Asp222] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mechanism through which ras could confer tumorigenic and metastatic potential (i.e., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1).


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Components of the MAP3 kinase signaling pathways (e.g., gip2, Ras, and Raf) cause oncogenic transformation in their constitutively active forms (1) . Moreover, expression of activated ras can confer metastatic potential upon some cells (reviewed in Ref. 2 ). The purposes of this study were: (a) to begin to ascertain what the downstream effectors of ras transformation and ras-induced metastatic potential are; and (b) to use stable Mek1 mutant variants to address whether ERK1/2 activity is essential for these phenotypes.

Expression of constitutively active MEK1 in NIH3T3 fibroblasts results in cellular transformation (3) . Activation of MEK1 is accomplished by phosphorylation of serines at positions 218 (S218) and 222 (S222; Ref. 3 ). To create constitutively active MEK1, S218 and S222 were mutated to aspartic acid, mimicking the phosphorylated/active state. The MEK1-activated mutants were designated DS and DD, where DS is Asp218/Ser222 and DD is Asp218/Asp222. DS and DD clonal cell lines produced colonies when grown in soft agar, an in vitro indicator of transformation. However, anchorage-independent growth did not correlate with ERK1/2 activity. The DS (DS2 and DS4) lines exhibited constitutively active ERK1/2, yet yielded fewer colonies compared with DD lines (DD1 and DD3), which had basal ERK1/2 activity. These data suggested that maintenance of transformation was independent of ERK1/2 activity.

Recently, Webb et al. (4) , using various ras mutants, showed that although tumorigenicity was independent of ERK1/2 activity, metastasis required its activation. Therefore, we wanted to determine whether clonal cell lines that we established previously and that exhibited constitutive or basal levels of ERK1/2 activity could also confer tumorigenicity and/or metastatic potential. Our data show that tumorigenic and metastatic potentials are dependent upon MEK1 activation but appear to be independent of ERK1/2 activity.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Analysis of Metastatic Potential of MEK1-transformed Clonal Lines.
Single-cell suspensions of DS and DD were made in ice-cold HBSS and were injected into the lateral tail veins of female athymic mice, 3–4 weeks of age, in a total volume of 0.2 ml/mouse. Mice were killed by methoxyflurane (Metofane; Pitman-Moore, Washington Crossing, NJ), followed by cervical dislocation. Complete necropsies were performed, and metastases were quantified as described (5) . Lung metastases were counted after fixing whole lungs in a mixture of neutral buffered formalin and Bouin’s fixative (5:1). Random 4–6-µm H&E-stained sections were examined. All animal studies were performed according to the guidelines of the NIH, and protocols were approved by the Institutional Animal Care and Use Committee.

Western Blot.
Lungs were isolated 27–41 days after i.v. inoculation from NIH3T3-, DS- and DD-injected athymic mice. The tissue was analyzed by Western blotting as described (3) with some modifications. Briefly, lungs were lysed by Dounce homogenization in potassium phosphate buffer [10 mM KPO4 (pH 7.05), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM ß-glycerophosphate, 1 mM sodium vanadate, 1 mM DTT, 0.5% NP40, 0.1% Brij-3, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A]. Lung metastases were dissected from adjacent normal tissues before further processing. Lysates were centrifuged at 16,000 x g for 10 min. Supernatants (40 µg) were boiled in 1x sample buffer [500 mM Tris-HCl (pH 6.8), 10% SDS, 20% glycerol, 0.05% bromphenol blue, and 1% 2-mercaptoethanol] for 5 min and electrophoresed on a 10% SDS polyacrylamide gel. Proteins were then transferred to polyvinylidene difluoride Immobilon membrane (Millipore, MA) and probed with phosphospecific ERK1/2 antibody (New England Biolabs, Beverly, MA) at a dilution of 1:1000 at 4°C overnight in PBS containing 0.1% Tween 20 and 3% BSA. Membranes were then incubated with horseradish peroxidase-conjugated antirabbit antibody (Amersham) at a dilution of 1:2000 at room temperature for 20 min, and the signal was detected using electrochemiluminescence (ECL; Amersham), followed by exposure to X-OMAT AR film (Eastman Kodak, Rochester, NY). Blots were stripped and reblotted with anti-ERK1/2 antibody (C-14; Santa Cruz Biotechnology, Biotechnology, CA) to determine equal loading of samples. Stripping was accomplished by submerging the membrane in 100 mM 2-mercaptoethanol, 20% SDS, and 62.5 mM Tris-HCl (pH 6.7) for 30 min at 55°C, followed by washing two times in PBS/0.1% Tween 20 for 10 min.

Growth in Soft Agar and on Bacterial Petri Plates.
Cells (1 x 105) were plated onto 60-mm bacterial Petri plates (Fisher Scientific, Pittsburgh, PA) or tissue culture plates (Corning, Oneata, NY) and examined daily for growth and proliferation by counting total cell number using a hemacytometer. The methods used for assessing growth on bacterial plates were identical to those of Rieber et al. (6) . Growth in soft agar was done as described (7 , 8) using 0.25% agar.

Gelatin Enzymography.
Enzymography was done by seeding cells (1.5 x 106/well) in a 12-well plate, followed by incubation at 37°C for 24–48 h. The complete medium was then removed and replaced with serum-free medium, and the cells were incubated at 37°C for 24 h. The next day, the supernatant was removed and spun at 1500 rpm for 10 min. Samples were solubilized in electrophoresis sample buffer containing SDS, absent of ß-mercaptoethanol. Samples, normalized to volume and cell number, were loaded onto a 7.5% SDS-PAGE gel containing 1 mg/ml gelatin. After the gel was run, it was transferred to Triton X-100 and incubated for 1 h at room temperature. It was then incubated in reaction buffer for 24 h at 37°C. The gel was stained with Coomassie Blue and destained, and the MMP2 and MMP9 bands were visualized.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
MEK1 Mutant Clones Grow on Adhesion-restricted Substrates.
DS and DD clones were shown to exhibit differential ERK1/2 activation (Fig. 1)Citation . ERK activation levels are consistent with those presented (3) . Also, we had shown previously that DS and DD clones produced transformed foci on tissue culture plastic and also produced multicellular colonies in soft agar. Both properties have been correlated with transformation. Recently, Rieber et al. (6) developed an assay in which melanoma cells are seeded onto bacterial Petri dishes. Whereas normal melanocytes undergo anoikis under these conditions, tumorigenic cells form spheroid-like masses of non- or poorly proliferating cells. In contrast, metastatic melanoma cell lines adhere to the dishes and proliferate (6) . We adapted this assay for use with the MEK1 clonal variants.



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  		Fig. 1. Differential activation of ERK1/2 by constitutively active MEK1 mutants. Western blot analysis of lysates (40 µg) from MEK1-DS, MEK1-DD, and NIH3T3 cells and lung metastases was performed with phosphospecific-ERK1/2 antibodies (ERK-P). The blot was stripped and reprobed with antibody to total ERK1/2 protein (ERK) to assess equal loading of lysates.

 
NIH3T3, DS, and DD cells survived after seeding onto the bacterial plates. All of the cell lines tended to form spheroid-like structures on the bacterial Petri dishes but were, as expected, spread and exhibited characteristic fibroblast-like morphology on tissue culture plastics (data not shown). NIH3T3 cells, which are immortal but not tumorigenic, divided once on the bacterial substrate. DS2 and DS4 cells underwent between 2 and 3 cell divisions within 1 week after seeding, whereas DD1 and DD3 cells grew at rates similar to those observed on tissue culture plastic (Fig. 2)Citation . Both DD1 and DD3 cells experienced a lag before rapid, exponential growth. We have yet to determine the reasons for this lag, although it is possible that the cells are providing a matrix upon which proliferation can be facilitated.



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  		Fig. 2. Growth rates of MEK1 clonal lines on bacterial versus tissue culture plates. A, cells were grown on bacterial ({circ}) and tissue culture (•) dishes and photographed after plating using a Nikon Diaphot microscope at x40. B, cells were counted at the days indicated. Numbers represent the values from three wells; bars, SE.

 
MEK1 Mutant Clones Are Tumorigenic.
On the basis of the in vitro transformation phenotypes, DS and DD variants were analyzed for their ability to form tumors after s.c. injection into female athymic mice. Tumors grew rapidly and progressively, detectable within 7–14 days after inoculation with 1 x 106 cells. Parental NIH3T3 cells failed to produce tumors after injection into mice.

DS variant clones exhibited constitutively active ERK1/2 activity whereas the DD clones have basal levels, yet both were equally tumorigenic. This result implies that tumorigenicity is independent of ERK activity. This conclusion is also suggested by Webb et al. (4) , who used ras transformants.

DD and DS Variants Are Metastatic.
Each of the clonal cell lines expressing constitutively active MEK1 formed metastases after i.v. injection into the tail veins of athymic mice. Table 1Citation shows data from two independent experiments in which the MEK1 clonal lines aggressively colonized mouse lungs. These results corroborated and extended previous studies that showed that activated ras could confer tumorigenic and metastatic potentials upon NIH3T3 cells (2) . Indeed, it showed that at least some portion of these phenotypes were mediated through the MAP kinase pathway downstream of MEK1.


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  		Table 1 MEK1 mutants are metastatic after i.v. inoculation into athymic mice

Results are shown from two independent experiments. Single-cell suspensions of cells in ice-cold HBSS were injected into the lateral tail vein of 3–4-week-old female athymic mice in a total volume of 0.2 ml/mouse. Upon killing by anesthesia with Metofane followed by cervical dislocation, complete necropsies were done. Metastases were found only in the lungs. To facilitate quantification, lungs were placed into a solution of neutral-buffered formalin and Bouin’s fixative (1:5), as described previously (5). Experiment 1 was terminated 41 days after injection. For experiment 2, mice were killed 27 days after injection.

 
On average, DD metastases were slightly but not significantly smaller than DS lesions. In experiment 1, i.v. injection of 5.4 x 105 DD or DS cells produced >250 lung metastases/mouse. In the second experiment, the number of cells injected per mouse was reduced to 3 x 105, and the length of the experiment was shortened (27 days instead of 41 days) to discriminate potential differences more easily. DS2 and DS4 clones that exhibited constitutively active endogenous ERK1/2 (Fig. 1)Citation yielded an average of 28 and 47 lung metastases, respectively (Table 1)Citation . DD1 and DD3, which exhibit only basal ERK1/2 activity when compared with NIH3T3 cells (Fig. 1)Citation , produced an average of 5 and >250 lung metastases/mouse, respectively. Other organs did not support metastatic growth, as determined by gross observation and random histological sections. Parental NIH3T3 cells did not develop metastatic nodules in any other tissues. These data suggest that the metastatic potential to lung of the MEK1 clonal lines is independent of initial ERK activity. Metastases resulting from DS and DD clones exhibited characteristics consistent with highly invasive and poorly differentiated fibrosarcoma (Fig. 3A)Citation .



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  		Fig. 3. MEK1 clonal lines confer metastatic potential in mice. A, histological appearance of lung metastases from mice that were i.v.-injected with MEK1 clonal lines show the appearance fibrosarcomas. H&E-stained cross sections of the lungs are shown. x150. The enhancement of metastasis may be attributable to increased MMP2 and MMP9 activity in MEK1 clonal lines. B, enzymography using gelatin as a substrate was performed using equal loading on a per cell basis.

 
Development and/or Maintenance of DS and DD Metastases Does Not Require ERK Activity.
Phospho-ERK1/2 activity was measured in the lung metastases isolated from mice in experiment 2. For the DD and DS variants, metastases were dissected from adjacent normal tissue to minimize the impact of activities within the stroma when interpreting the results. Dissection was particularly easy for the DD3 metastases, some of which were >3 mm in diameter. The left set of lanes in Fig. 1Citation shows that lung metastases isolated from DS2- and DS4-injected mice still exhibited both ERK1 and ERK2 phosphorylation. In contrast, DD1-injected mice exhibited phosphorylation of ERK1, but DD3-injected mice, which yielded the highest number of lung metastases (e.g., >250), showed no detectable ERK1 phosphorylation, similar to that observed in the lungs of NIH3T3-injected mice. The data suggest that lung colonization is independent of ERK activity. This finding does not eliminate the possibility that ERK is transiently activated during transport in the circulation and/or early colonization.

DS and DD Clones Show Increased MMP2 and MMP9 Activity.
Using gelatin enzymography, we demonstrated that the MEK1-transformed clonal lines also show an increase in the levels of MMP2 and MMP9 (Fig. 3B)Citation , two MMPs that have been implicated in metastasis (9) . SDS-PAGE of cell culture supernatants from DS and DD variants also revealed increased levels of a Mr ~39,000 protein. Isolation and sequencing of this protein revealed that it was cathepsin L.4 Therefore, it is possible that MEK1 transformation leads to the production of proteinases, such as MMP2, MMP9, and cathepsin L, that contribute to metastatic potential.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The molecular basis for cancer metastasis has not been fully elucidated. It requires the coordinated expression of multiple genes so that cells migrate from the primary tumor mass, enter a circulatory system, survive transport, arrest at a secondary site, and respond to proliferation signals at the secondary site. Both positive and negative regulatory effectors have been identified (reviewed in Ref. 10 ).

Previous studies have demonstrated that introduction of oncogenic forms of the ras oncogene confer both tumorigenic and metastatic potentials upon NIH3T3 cells. These results implicated events downstream in the regulation of metastasis. The current study was to begin identifying key downstream components contributing to tumorigenicity and metastasis. Toward that end, constitutively active variants of MEK1, a downstream component of the ras/MAPK pathway that has been shown previously to lead to cellular transformation, were used. Like ras, MEK1 transformants of NIH3T3 are not only tumorigenic but are also metastatic. Thus, our results refine the mechanism through which ras can confer these phenotypes and imply that the critical determinants are downstream of MEK1.

Recently, Webb et al. (4) used ras transfectants to show that tumorigenicity occurred through both Raf-dependent and -independent pathways. In contrast, metastatic potential in their model correlated with variants that were able to activate ERK1 activity. Seven to 9 weeks after injection into mice, some of the ras transfectants (that were originally nonmetastatic) formed lung colonies. This implies that some selection may have occurred, and the authors suggest that increased expression of the Met receptor tyrosine kinase is responsible, although only modest increases in ERK1/2 activity was observed in these variants. Hepatocyte growth factor, the ligand of the Met receptor, can induce both the MEK1 and phosphatidylinositol 3-kinase pathways, leaving the possibility that the pathway responsible for acquisition of metastasis in these cells is MEK1 and/or phosphatidylinositol 3-kinase dependent.

Although we are in agreement with Webb et al. (4) concerning tumorigenicity and how the process may be ERK1/2 independent, we have contrasting results regarding metastatic potential and ERK1/2 activity. This discrepancy may be attributable to the fact that we specifically analyzed MEK1 transformants and not ras transformants. It is also possible that MEK1-induced transformation may occur through a pathway that is distinct from that of ras transformation and may not necessarily require ERK1/2 activity to maintain transformation.

The current results are consistent with those in which we showed previously that growth in soft agar is independent of ERK activity. DS and DD have varying ERK activity, and we wanted to know whether, in fact, metastasis was dependent upon the level of ERK activity. Western blot data of the lung metastases show that, in fact, metastatic potential is independent of ERK activity, suggesting that the signaling pathway diverges downstream of MEK1, activating as yet undefined components. Although the downstream components have yet to be identified, we do know that these clonal cell lines express and secrete the proteinases MMP2, MMP9, and cathepsin L.

Expression of MMPs has been correlated with metastatic potential as well as ERK, c-Jun NH2 kinase, and p38 kinase pathways (11) , although it was shown previously that stimulation of the MMP9 promoter by ras is independent of MEK1, requiring multiple transcription factor binding sites. Recently, McCawley et al. (12) have shown that sustained activation of ERK led to increased MMP-9 activity and cell migration in keratinocytes.

Likewise, increased expression of cathepsin L has been correlated with metastatic potential in ras-transfected cell lines (13) . As for the MMPs, higher expression and activity of cathepsin L could lead to higher efficiency penetration of physiological barriers or increased cathepsin L can be involved in tumor cell evasion of immune response by cleavage of the third component of complement (14) .

In conclusion, the results presented here refine one pathway involved in the regulation of metastasis by demonstrating that mediators downstream of MEK1 are involved. Moreover, the results imply that ERK1/2 activity are not essential for the development and/or maintenance of metastatic foci.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grants CA62168 (to D. R. W.), NS10828 (to A. A. and J. V. B.), DK39773 (to J. V. B.) from the NIH; the United States Army Medical Research and Materiel Command DAMD17-96-1-6152 (to D. R. W.); the National Foundation for Cancer Research (to D. R. W.); The American Heart Association—Massachusetts Division; and CONICIT grants S1-96001340 and G-9700613 (to M. R. and M. S. R.). Back

2 To whom requests for reprints should be addressed, D. R. W. at Jake Gittlen Cancer Research Institute, Room C7810, Box H-059, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033-2390 or A. A. at Renal Unit, Massachusetts General Hospital-East, 149 13th Street, Charlestown, MA 02129. Back

3 The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; DS, MEK1-DS; DD, MEK1-DD; MMP, matrix metalloproteinase. Back

4 Q. Hon, A. Alessandrini, and R. Erikson, unpublished data. Back

Received 12/13/99. Accepted 2/ 2/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Campbell S. L., Khosravi-Far R., Rossman K. L., Clark G. J., Der C. J. Increasing complexity of Ras signaling. Oncogene, 17: 1395-1413, 1998.[Medline]
  2. Chambers A. F., Tuck A. B. Ras-responsive genes and tumor metastasis. Crit. Rev. Oncog., 4: 95-114, 1993.[Medline]
  3. Alessandrini A., Greulich H., Huang W., Erikson R. L. Mek1 phosphorylation site mutants activate Raf-1 in NIH 3T3 cells. J. Biol. Chem., 271: 31612-31618, 1996.[Abstract/Free Full Text]
  4. Webb C. P., Van Aelst L., Wigler M. H., Vande W. G. Signaling pathways in Ras-mediated tumorigenicity and metastasis. Proc. Natl. Acad. Sci. USA, 95: 8773-8778, 1998.[Abstract/Free Full Text]
  5. Welch D. R. Technical considerations for studying cancer metastasis in vivo. Clin. Exp. Metastasis, 15: 272-306, 1997.[Medline]
  6. Rieber M., Welch D. R., Strasberg-Rieber M. Differential gene expression associated with suppression of metastatic melanoma cell spreading and proliferation on adhesion-restrictive substrates. Proc. Am. Assoc. Cancer Res., 40: 491 1999.
  7. Lee J-H., Welch D. R. Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1. Cancer Res., 57: 2384-2387, 1997.[Abstract/Free Full Text]
  8. Nicolson G. L., Lembo T. M., Welch D. R. Growth of rat mammary adenocarcinoma cells in semisolid clonogenic medium not correlated with spontaneous metastatic behavior: heterogeneity in the metastatic, antigenic, enzymatic and drug sensitivity properties of cells from different sized colonies. Cancer Res., 48: 399-404, 1988.[Abstract/Free Full Text]
  9. Chambers A. F., Matrisian L. M. Changing views of the role of matrix metalloproteinases in metastasis. J. Natl. Cancer Inst., 89: 1260-1270, 1997.[Abstract/Free Full Text]
  10. Welch D. R., Wei L. L. Genetic and epigenetic regulation of human breast cancer progression and metastasis. Endocr. Relat. Cancer, 5: 155-197, 1998.[Abstract]
  11. Ghiso J. A., Alonso D. F., Farias E. F., Gomez D. E., Kier Joffe E. B. Deregulation of the signaling pathways controlling urokinase production. Its relationship with the invasive phenotype. Eur. J. Biochem., 263: 295-304, 1999.[Medline]
  12. McCawley L. J., Li S., Wattenberg E. V., Hudson L. G. Sustained activation of the mitogen-activated protein kinase pathway. A mechanism underlying receptor tyrosine kinase specificity for matrix metalloproteinase-9 induction and cell migration. J. Biol. Chem., 274: 4347-4353, 1999.[Abstract/Free Full Text]
  13. Chambers A. F., Colella R., Denhardt D. T., Wilson S. M. Increased expression of cathepsins L and B and decreased activity of their inhibitors in metastatic, ras-transformed NIH 3T3 cells. Mol. Carcinog., 5: 238-245, 1992.[Medline]
  14. Frade R., Rodrigues-Lima F., Huang S. Y., Xie K. P., Guillaume N., Bar-Eli M. Procathepsin-L, a proteinase that cleaves human C3 (the third component of complement), confers high tumorigenic and metastatic properties to human melanoma cells. Cancer Res., 58: 2733-2736, 1998.[Abstract/Free Full Text]



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O. P. Barry, B. Mullan, D. Sheehan, M. G. Kazanietz, F. Shanahan, J. K. Collins, and G. C. O'Sullivan
Constitutive ERK1/2 Activation in Esophagogastric Rib Bone Marrow Micrometastatic Cells Is MEK-independent
J. Biol. Chem., April 27, 2001; 276(18): 15537 - 15546.
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