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Tumor Biology |
Department of Internal Medicine, Division of Hematology and Oncology and Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Until recently, relatively little was known about the genetic mechanisms underlying the development and progression of IBC. In a previous study, our laboratory identified genes that strongly correlated with the aggressive and invasive IBC phenotype (4) . In this study, we describe the potential role of one of those genes, RhoC GTPase, in modulating the invasive IBC phenotype.
RhoC GTPase is a member of the Ras superfamily of small GTPases. Activation of Rho proteins by soluble factors, such as serum or growth factors, leads to the assembly of actin-myosin contractile filaments and focal adhesion complexes (5 , 6) . "Cross-talk" interactions between the various members of the Ras superfamily lead to the dynamic reorganization of the cell cytoskeleton, resulting in polarity, the formation of lamellipodia, and adhesion during directed motility (7) .
We hypothesized that, given these known functions of the Rho proteins, RhoC GTPase would confer to mammary epithelial cells specific features of enhanced invasiveness and tumorigenic potential. To test this hypothesis, we set out to determine whether overexpression of RhoC GTPase could alter the phenotype of nontransformed, immortalized HME cells. We generated stable HME-RhoC transfectants and tested them for any alterations in their phenotype. In contrast with either untransfected or HME-ß-gal control, the HME-RhoC cells were able to form colonies under anchorage-independent growth conditions. Furthermore, these cells were highly invasive in a Matrigel invasion assay, exhibited enhanced motility in a random motility assay, and were tumorigenic in nude mice. These characteristics of the RhoC transfectants strikingly resemble in vivo behavior of IBC.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transfection Experiments.
Low passage (passage 10) immortalized HME cells were cotransfected with
the PTP-2000-hygro vector and either pFLAG-RhoC GTPase, pFLAG-ß-gal,
or pFLAG-CMV-4 (Sigma Chemical Co.) alone using FuGene 6 transfection
reagent (Roche-Boehringer Mannheim, Mannheim, Germany). Transfection
efficiency was 22–85% as determined by ß-gal staining of the
pFLAG-ß-gal-transfected cells using X-Gal reagent [1 mg/ml X-Gal, 4
mM potassium ferricyanide, 4 mM potassium
ferrocyanide, 2 mM magnesium chloride-hexahydrate in PBS
(pH 7.4); Life Technologies, Inc., Gaithersburg, MD] and 2%
formaldehyde and 0.2% glutaraldehyde in cation-free PBS [(pH 7.4),
Sigma Chemical Co.] for 16 h at 37°C. Stable transfectants were
established by culturing the cells in the described medium supplemented
with 100 µg/ml hygromycin (Life Technologies, Inc.) for 14–20 days.
Expression of the transgene was determined by RT-PCR and
immunoprecipitation followed by Western blot analysis.
Constructs.
RhoC GTPase from the SUM149 cell line was amplified by RT-PCR and
cloned using the pGEM-T Easy kit (Promega, Madison, WI). The
full-length RhoC GTPase cDNA was sequenced and checked against the
published sequence of any mutations. Using EcoRI
(Roche-Boehringer Mannheim), the RhoC cDNA was excised and ligated into
pFLAG-CMV-4 (Sigma Chemical Co.) and resequenced.
Expression Analysis of RhoC GTPase Transfectants.
Total RNA was isolated from cells using Trizol reagent (Life
Technologies, Inc.) according to the manufacturer’s recommendations.
One µg of total RNA was converted to cDNA using an avian
myeloblastosis virus reverse transcription system (Promega). A 100-µg
aliquot of the resulting cDNA was amplified in a double PCR with 25 ng
each of pFLAG/GAPDH- or RhoC GTPase/GAPDH-specific primers. PCR
products were separated on a 1.2% agarose gel and imaged on an Alpha
Image 950 documentation system (Alpha Innotech, San Leandro, CA).
Densitometry of images was performed using NIH Image version 1.62.
Proteins were harvested from cell cultures using radioimmunoprecipitation assay buffer (1x PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 0.3 mg/ml aprotinin; Sigma Chemical Co.). Whole cell lysates (100 µg) were precleared using normal mouse IgG and protein A/G-agarose (Santa Cruz Biotechnology Inc., Santa Cruz, CA). The cleared supernatants were incubated overnight with antibodies specific for the FLAG epitope (Eastman Kodak, Rochester, NY). Antibody-bound proteins were precipitated after the addition of protein A/G-agarose and washed four times with 1x PBS. Immunoprecipitates were resuspended in 20 µl of 2x Laemmli electrophoresis buffer, boiled for 3 min, and centrifuged briefly to pellet the free protein A/G-agarose. Samples were separated by SDS-PAGE on an 8% gel under nondenaturing conditions and transferred to a nitrocellulose membrane. Nonspecific binding was blocked by overnight incubation with 2% powdered milk (Kroger, Cincinnati, OH) in Tris-buffered saline with 0.05% Tween 20 (Sigma Chemical Co.). Anti-FLAG immunoprecipitate blots were incubated with the FLAG antibody. Protein bands were visualized by ECL (Amersham-Pharmacia Biotech, Piscataway, NJ).
Monolayer Growth Rate.
Monolayer culture growth rate was determined as described previously
(12)
by conversion of MTT (Sigma Chemical Co.) to a
water-insoluble formazan by viable cells. Three thousand cells in 200
µl of medium were plated in 96-well plates and grown under normal
conditions. Cultures were assayed at 0, 1, 2, 3, 5, and 7 days by the
addition of 40 µl of 5 mg/ml MTT and incubation for 1 h at
37°C. The MTT-containing medium was aspirated, and 100 µl of DMSO
(Sigma Chemical Co.) were added to lyse the cells and solubilize the
formazon. Absorbance values of the lysates were determined on a
Dynatech MR 5000 microplate reader at 540 nm.
Anchorage-independent Growth in Soft Agar.
A 2% stock of sterile, low melting point agarose was diluted
1:1 with 2x MEM. Further dilution to 0.6% agarose was made using 10%
FBS-supplemented Ham’s F-12 medium complete with growth factors, and 1
ml was added to each well of a 6-well plate as a base layer. The cell
layer was then prepared by diluting agarose to 0.3% and 0.6% with
103
cells in 2.5% FBS-supplemented Ham’s F-12
medium/1.5 ml/well. Colonies greater than or equal to 100
µM in diameter were counted after a 3-week incubation at
37°C in a 10% CO2 incubator.
Invasion through a Matrigel-coated Filter.
The invasion assay was performed as described previously, with minor
modifications (12)
. A 10-µl aliquot of Matrigel (10
mg/ml) was spread onto a 6.5-mm Transwell filter with 8 µm pores
(Costar, Corning, NY) and air dried in a laminar flow hood. Once dried,
the filters were reconstituted with a few drops of serum-free medium.
The lower chamber of the Transwell was filled with either serum-free or
serum-containing media. Cells were harvested and resuspended in
serum-free medium with 0.1% BSA at a concentration of 3.75 x 105 cells/ml, and 0.5 ml of cell
suspension was added to the top chamber. The chambers were
incubated for 24 h at 37°C in a 10% CO2
incubator. The cell suspension was aspirated, and excess Matrigel was
removed from the filter using a cotton swab. The filters were then cut
away from the Transwell assembly and fixed with methanol, gel side
down, to a glass microscope slide. The fixed filters were stained with
H&E, and the cells in 20 random x40 magnification fields counted.
These cells were assumed to have invaded through the Matrigel/filter.
The number of cells that had invaded the serum-free containing lower
chambers was considered background, and this number was subtracted from
the number of cells that had invaded in response to the
serum-containing medium.
Random Motility Assay.
Random motility was determined using a gold-colloid assay
(13)
. Gold-colloid was layered onto glass coverslips and
placed into 6-well plates. Cells were plated onto the coverslips and
allowed to adhere for 1 h at 37°C in a CO2
incubator (12,500 cells/3 ml in serum-free medium). To stimulate the
cells, the serum-free medium was replaced with 5% FBS-containing
Ham’s F-12 medium supplemented with growth factors and allowed to
incubate for 3 h at 37°C. The medium was aspirated, and the
cells were fixed using 2% glutaraldehyde. The coverslips were then
mounted onto glass microscope slides, and areas of clearing in the
gold-colloid corresponding to phagokinetic cell tracks were counted.
Rhodamine-Phalloidin Staining of Actin Filaments.
Visualization of actin filaments was accomplished by staining with a
conjugated phallotoxin. Cells were grown on glass coverslips for
48 h and washed with PBS followed by fixation with 1:1 ice-cold
acetone and methanol. After a 30-min incubation in PBS containing 1%
BSA, 5 µl of methanolic rhodamine-phalloidin stock (Molecular Probes,
Eugene, OR) were added to each coverslip and allowed to stain for 20
min at room temperature. After repeated washing with PBS, the
coverslips were mounted onto glass microscope slides using Gel/Mount
(Biomedia Co., Foster City, CA). Cells were visualized under an Olympus
fluorescence microscope equipped with a 573 nm filter.
Orthotopic Injection into Nude Mice.
Ten-week-old female NCR athymic nude mice were orthotopically injected
with HME-, HME-ß-gal-, or HME-RhoC GTPase-transfected cells.
Injection into the MFP of 20 mice/group was performed as described
previously (14)
. Briefly, cells were harvested by
trypsinization, washed, and resuspended in HBSS at a concentration of
1.5 x 106 cells/0.2 ml. Mice were
anesthetized using 10 mg/ml ketamine, 1 mg/ml xylazine, and 0.01 mg/ml
glycopyrrolate, and an incision below the thoracic MFP was made. Using
a 27-gauge needle, the cell suspension was injected directly into the
exposed MFP, and the wound was closed with a single wound clip. Tumor
growth measurements were taken weekly until tumors reached 2.5 cm in
diameter. Mice were necropsied, and tumor tissues were dissected for
histological analysis.
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RESULTS |
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. Levels of RhoC GTPase expression were found to be
6–20-fold higher in RhoC GTPase transfectants when compared with the
untransfected or HME-ß-gal control-transfected HME cells. This
level of expression is comparable with that of the SUM149 IBC cell
line, which was found to have an 8-fold higher expression of
RhoC as compared with normal HME cell lines and non-IBC
breast cancer cell lines as determined by Northern blot analysis
(4)
. Also, this level of expression was comparable to
archival IBC tumor specimens, which were previously found to have
5–12-fold higher levels of RhoC expression compared with
the surrounding normal tissue as determined by in situ
hybridization (4)
. Expression of the FLAG-tagged RhoC
GTPase protein was confirmed by immunoprecipitation with an anti-FLAG
antibody followed by immunoblotting with the same antibody (Fig. 1B)
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, no significant changes in the monolayer growth rates were
observed for any of the HME-RhoC transfectant clones or the mixed
polyclonal population, as compared with the control cell lines using
the MTT assay. Furthermore, regardless of quantitative differences in
the level of RhoC transgene expression, no difference in growth rate
was observed between the various clones and the polyclonal population.
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the number of colonies greater than 100 µm in size formed by
individual HME-RhoC clones ranged from 6–176 colonies/well in
comparison to the HME-ß-gal cells, which had a range of 0–6
colonies/well. This increase in anchorage-independent growth directly
correlated with levels of expression of the RhoC transgene. The
polyclonal population had the highest level of colony formation, and
this recapitulated the behavior of the SUM149 IBC cell line in soft
agar. The SUM149 cell line expressed RhoC GTPase at 8-fold higher
levels than the HMEs. In the same assay, using the less stringent
condition of 0.3% soft agar, the average HME-RhoC transfectant
colony size was greater than 600 µm.
Overexpression of RhoC GTPase Produces a Motile and Invasive
Phenotype in Immortalized HME Cells through Formation of Actin Stress
Fibers and Focal Adhesion Points.
The term IBC was coined in 1924 to describe the appearance of the skin
overlaying the affected breast (16)
. This term is somewhat
of a misnomer because IBC is not characterized by significant
infiltration of inflammatory cells. The inflamed appearance of the skin
is due to edema caused by invasion into and obstruction of the dermal
lymphatics by tumor emboli (3)
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Rho proteins are most noted for their ability to control polarity, protrusion, and adhesion during cell motility by rearranging the actin cytoskeleton (6) . Given the clinical observations, coupled with the finding that RhoC GTPase is overexpressed in patient tumor samples (4) , we sought to ascertain whether RhoC GTPase overexpression could generate highly invasive and motile HME cells.
Using a modified blind-well chamber model, we measured the amount of
migration of cells across a porous membrane coated with the
extracellular matrix conglomerate Matrigel in response to a
chemoattractant. In this assay, all cell lines tested were invasive to
varying degrees after 24 h. The data in Fig. 3
are expressed as the fold increase in invasion observed over the
invasion level of the untransfected HME cell line. The HME-ß-gal
control-transfected cell line was 1.5-fold more invasive than the
untransfected cells, whereas the HME-RhoC transfectants were, on
average, 5-fold more invasive than either the untransfected or
HME-ß-gal cell lines. In comparison, the SUM149 primary IBC cell line
was 4-fold more invasive than the control (untransfected and ß-gal)
HME cell lines. As was observed previously in the anchorage-independent
growth assays, the ability to invade directly correlated with the level
of RhoC GTPase expression of the HME-RhoC transfectant clones. Invasion
rates from 1–19-fold over the control cell lines were observed,
depending on the RhoC transgene expression levels. These data
demonstrate that expression of RhoC GTPase directly correlates with the
ability of the cells to invade, with a clear threshold effect dependent
on RhoC expression level.
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depicts representative photomicrographs of a gold-colloid
random motility assay comparing HME-ß-gal and HME-RhoC transfectants.
In this assay, cells are layered onto a gold-colloid, on which motile
cells produce tracks that can then be quantitated. The data depicted in
Fig. 4B
are represented as a percentage of the total number
of cells of each clone that moved in response to serum activation over
a 3-h period. In this assay, we found that more than 90% of the
HME-RhoC cells were highly motile in response to serum stimulation.
This was true for the different HME-RhoC GTPase clones. In comparison,
only 10% of the HME-ß-gal control-transfected HME cells were motile
under the same conditions. Furthermore, in this assay, none of the
untransfected HME cells were motile.
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, focal adhesion points and actin stress
fibers are clearly visible and abundant in the HME-RhoC transfectants.
The same was true for the SUM149 IBC cell line (data not shown). These
prominent structures were not seen in the untransfected HME cells (data
not shown) or in HME-ß-gal control-transfected cells (Fig. 5A)
. The HME-RhoC clone 3 cell highlighted in Fig. 5B
clearly shows actin stress fiber formation emanating from
a focal adhesion point and leading to a discernible lamellipodial
extension. Taken together, these features imply much greater motility
and ability to effect cytoskeletal reorganization in the RhoC
transfectants than control HMEs.
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, none of the untransfected HME cells or HME-ß-gal cells formed tumors
in nude mice. Untransfected HME cells have never been known to form
tumors in nude mice. In contrast, both the SUM149 IBC cell line and the
RhoC GTPase HME cells grew tumors in nude mice, in 86% (17 of 20) and
25% (5 of 20) of cases, respectively. The incidence of tumor growth by
the SUM149 cell line is comparable with the SUM190 cell line, the only
other known available IBC cell line (data not shown). These data
indicate that overexpression of RhoC GTPase is weakly sufficient for
malignant transformation of immortalized HME cells and that other
genetic abnormalities in the IBC cell lines derived from
established tumors contribute to the full-blown malignant
phenotype.
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DISCUSSION |
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The mammalian Rho GTPase family can be divided in six different groups with the following members: (a) Rho (RhoA, RhoB, and RhoC); (b) Rac (RhoG and Rac1–3); (c) Cdc42 (Cdc42Hs, G25K, and TC10); (d) Rnd (Rho6, Rho7, and RhoE); (e) RhoD; and (f) TTF (19) . RhoA, RhoB, and RhoC share a high degree of homology with one another, with RhoA and RhoC being the most homologous (20) . Studies performed on Swiss 3T3 cells have helped to define the role that each of the Rho GTPase family members plays in controlling cytoskeletal reorganization (21) . Rho controls actin stress fiber and focal adhesion contact formation, whereas rac and Cdc42 are responsible for the formation of lamellipodia and filopodia, respectively (17) . Several upstream pathways that activate Rho as well as the downstream targets of activated Rho have been identified (15 , 22, 23, 24, 25) . The ability of Rho to form stress fibers and focal adhesions is due to the phosphorylation and activation of two of these targets, Rho-kinase/ROK/ROCK (24 , 26 , 27) and the myosin-binding subunit of myosin phosphatase (18 , 28) . Phoshorylation of myosin-binding subunit by Rho inhibits myosin phosphatase, whereas activated Rho-kinase directly phosphorylates the myosin light chain. The phosphorylation of myosin light chain regulates the formation of stress fibers and focal adhesion contacts. Rho-kinase can also phosphorylate adducin, which leads to cell membrane ruffling and cell motility (29) . It is also becoming clear that the various members of the Rho family (Rho, rac, and Cdc42) are able to undergo molecular "cross-talk," creating dynamic molecular interactions leading to cell motility (30 , 31) .
There is increasing evidence that the effects of Rho family members are, in fact, cell type specific and can extend beyond the actin cytoskeleton (reviewed in Ref. 6 ). For example, in keratinocytes, activated Rho and rac are responsible for cadherin-based adherens junctions (32) . However, the opposite is true in Madin-Darby canine kidney epithelial cells, in which activated Rho and rac decrease cadherin expression (33) . These observations demonstrate that expression of activated Rho GTPase can affect the expression of molecules, such as cadherins, that are integral in signal transduction pathways, thereby influencing transcription of other downstream genes (34 , 35) . Transcription of other genes, including those influencing motility, can also be regulated directly through activation of the mitogen-activated protein kinase subfamilies c-Jun NH2-terminal kinase (35) , and p38 kinase (36) .
Our initial finding that RhoC was a key molecule in an invasive cancer such as IBC was consistent with observations of other groups in different tumor types (37) . Suwa et al. (37) established a correlation between RhoC GTPase overexpression and the progression and prognosis of aggressive ductal adenocarcinoma of the pancreas. Other studies have demonstrated that activated Rho is an integral component for mediating cell motility required for producing intrahepatic metastasis of hepatocellular carcinoma (39) .
Overexpression of RhoC GTPase in immortalized HME cells produced a striking phenotypic change that, for the most part, recapitulates that of the SUM149 IBC cell line. The extent of the phenotypic change appears to correlate with the level of RhoC expression in the various HME-RhoC clones. However, the highest expressers do not completely recapitulate the SUM149 cell line, suggesting that other genetic alterations in the IBC cell line contribute to the full-blown malignant phenotype of the IBC cell. Growth under anchorage-independent conditions was greatly enhanced in the RhoC transfectants. Almost all of the control untransfected or ß-gal-transfected HME cells either underwent apoptosis (as determined by morphological changes) or were cytostatic under the same conditions. In comparison with the control cells, the RhoC transfectants produced up to 100-fold more colonies, which approached 63% of the level of colony formation of the SUM149 IBC cell line. Similarly, the effect of RhoC on both random and directed motility reproduced that of the SUM149 phenotype. As seen with anchorage-independent growth, the level of expression of RhoC GTPase influenced the level of bulk motility. As expected, there appears to be a threshold effect at the cellular level; even at lower (but still increased) expression levels, stress fiber and focal adhesion contact formation are seen. Further work in our laboratory4 indicates that RhoC overexpression modulates the angiogenic potential of the transfectants.
Finally, RhoC-HME cells produced tumors in nude mice. As expected, however, the uptake was considerably lower than that of wild-type SUM149. This is likely due to the absence of many other genetic events that contribute to tumor growth in the RhoC-HME transfectants. To quantitatively reproduce the in vivo tumorigenic phenotype of the SUM149 cell line, other genes, such as Ras, p53, or antiapoptotic genes may need to be altered as well. Therefore, overexpression of RhoC GTPase represents a single but important component of HME transformation.
The set of experiments that test the potential oncogenicity of a gene vary according to cell type and study. However, in general, they comprise a battery of assays that test in vitro- and in vivo-specific qualities of the phenotypic behavior (40 , 41) . The experiments we chose for this work were guided by the specific IBC phenotypic characteristics of the human tumors and the IBC parent cell line (SUM149) as well as by the putative function of the Rho proteins. For this particular system, we conclude that all of our experiments credential RhoC GTPase as an oncogene in breast cancer, which, to a significant extent, recapitulates the invasive behavior of our IBC model system. In addition, given the specific role that RhoC plays in the invasive phenotype of IBC, interfering with its function suggests a new therapeutic target in a particularly challenging form of breast cancer. As the upstream and downstream signaling pathways are explored, these too may present new targets for therapeutic intervention.
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FOOTNOTES |
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1 Supported by NIH Grants R01 CA 77612 (to
S. D. M.) and 5T32 CA09537-16, a grant (to S. D. M.) and a
Postdoctoral Fellowship (to K. L. v. G.) from The Susan G. Komen
Breast Cancer Foundation, and a grant from the Blodgett-Butterworth
Research Foundation (to S. D. M.). K. L. v. G. and Z-F. W.
contributed equally to this project and are considered co-first
authors. 
2 To whom requests for reprints should be
addressed, at Department of Internal Medicine, University of Michigan,
7217 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0948.
3 The abbreviations used are: IBC, inflammatory
breast cancer; HME, human mammary epithelial; FBS, fetal bovine serum;
RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
ß-gal, ß-galactosidase; MFP, mammary fat pad.
Received 3/ 1/00. Accepted 8/18/00.
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C. G. Kleer, T. N. Teknos, M. Islam, B. Marcus, J. S.-J. Lee, Q. Pan, and S. D. Merajver RhoC GTPase Expression as a Potential Marker of Lymph Node Metastasis in Squamous Cell Carcinomas of the Head and Neck Clin. Cancer Res., August 1, 2006; 12(15): 4485 - 4490. [Abstract] [Full Text] [PDF]
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J. J. Fiordalisi, P. J. Keller, and A. D. Cox PRL tyrosine phosphatases regulate rho family GTPases to promote invasion and motility. Cancer Res., March 15, 2006; 66(6): 3153 - 3161. [Abstract] [Full Text] [PDF]
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L. Zhang, L. Chenwei, R. Mahmood, K. v. Golen, J. Greenson, G. Li, N. J. D'Silva, X. Li, C. F. Burant, C. D. Logsdon, et al. Identification of a Putative Tumor Suppressor Gene Rap1GAP in Pancreatic Cancer Cancer Res., January 15, 2006; 66(2): 898 - 906. [Abstract] [Full Text] [PDF]
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N. Hakuma, I. Kinoshita, Y. Shimizu, K. Yamazaki, K. Yoshida, M. Nishimura, and H. Dosaka-Akita E1AF/PEA3 Activates the Rho/Rho-Associated Kinase Pathway to Increase the Malignancy Potential of Non-Small-Cell Lung Cancer Cells Cancer Res., December 1, 2005; 65(23): 10776 - 10782. [Abstract] [Full Text] [PDF]
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A. C. Berzat, J. E. Buss, E. J. Chenette, C. A. Weinbaum, A. Shutes, C. J. Der, A. Minden, and A. D. Cox Transforming Activity of the Rho Family GTPase, Wrch-1, a Wnt-regulated Cdc42 Homolog, Is Dependent on a Novel Carboxyl-terminal Palmitoylation Motif J. Biol. Chem., September 23, 2005; 280(38): 33055 - 33065. [Abstract] [Full Text] [PDF]
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Q. Pan, L. W. Bao, C. G. Kleer, M. S. Sabel, K. A. Griffith, T. N. Teknos, and S. D. Merajver Protein Kinase C{varepsilon} Is a Predictive Biomarker of Aggressive Breast Cancer and a Validated Target for RNA Interference Anticancer Therapy Cancer Res., September 15, 2005; 65(18): 8366 - 8371. [Abstract] [Full Text] [PDF]
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 B. M. Jacobsen, S. A. Schittone, J. K. Richer, and K. B. Horwitz Progesterone-Independent Effects of Human Progesterone Receptors (PRs) in Estrogen Receptor-Positive Breast Cancer: PR Isoform-Specific Gene Regulation and Tumor Biology Mol. Endocrinol., March 1, 2005; 19(3): 574 - 587. [Abstract] [Full Text] [PDF]
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K. J. Simpson, A. S. Dugan, and A. M. Mercurio Functional Analysis of the Contribution of RhoA and RhoC GTPases to Invasive Breast Carcinoma Cancer Res., December 1, 2004; 64(23): 8694 - 8701. [Abstract] [Full Text] [PDF]
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N. Liu, F. Bi, Y. Pan, L. Sun, Y. Xue, Y. Shi, X. Yao, Y. Zheng, and D. Fan Reversal of the Malignant Phenotype of Gastric Cancer Cells by Inhibition of RhoA Expression and Activity Clin. Cancer Res., September 15, 2004; 10(18): 6239 - 6247. [Abstract] [Full Text] [PDF]
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G. Somlo, P. Frankel, W. Chow, L. Leong, K. Margolin, R. Morgan Jr, S. Shibata, P. Chu, S. Forman, D. Lim, et al. Prognostic Indicators and Survival in Patients With Stage IIIB Inflammatory Breast Carcinoma After Dose-Intense Chemotherapy J. Clin. Oncol., May 15, 2004; 22(10): 1839 - 1848. [Abstract] [Full Text] [PDF]
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J. Du, B. Jiang, R. J. Coffey, and J. Barnard Raf and RhoA Cooperate to Transform Intestinal Epithelial Cells and Induce Growth Resistance to Transforming Growth Factor {beta} Mol. Cancer Res., April 1, 2004; 2(4): 233 - 241. [Abstract] [Full Text] [PDF]
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 K. Wennerberg and C. J. Der Rho-family GTPases: it's not only Rac and Rho (and I like it) J. Cell Sci., March 15, 2004; 117(8): 1301 - 1312. [Abstract] [Full Text] [PDF]
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L. M. McHardy, R. Sinotte, A. Troussard, C. Sheldon, J. Church, D. E. Williams, R. J. Andersen, S. Dedhar, M. Roberge, and C. D. Roskelley The Tumor Invasion Inhibitor Dihydromotuporamine C Activates RHO, Remodels Stress Fibers and Focal Adhesions, and Stimulates Sodium-Proton Exchange Cancer Res., February 15, 2004; 64(4): 1468 - 1474. [Abstract] [Full Text] [PDF]
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M. Cristofanilli, E. S. Singletary, and G. N. Hortobagyi Inflammatory Breast Carcinoma: The Sphinx of Breast Cancer Research J. Clin. Oncol., January 15, 2004; 22(2): 381 - 383. [Full Text] [PDF]
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W. G. Jiang, G. Watkins, J. Lane, G. H. Cunnick, A. Douglas-Jones, K. Mokbel, and R. E. Mansel Prognostic Value of Rho GTPases and Rho Guanine Nucleotide Dissociation Inhibitors in Human Breast Cancers Clin. Cancer Res., December 15, 2003; 9(17): 6432 - 6440. [Abstract] [Full Text] [PDF]
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L. Wang, L. Yang, Y. Luo, and Y. Zheng A Novel Strategy for Specifically Down-regulating Individual Rho GTPase Activity in Tumor Cells J. Biol. Chem., November 7, 2003; 278(45): 44617 - 44625. [Abstract] [Full Text] [PDF]
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Y. Shikada, I. Yoshino, T. Okamoto, S. Fukuyama, T. Kameyama, and Y. Maehara Higher Expression of RhoC Is Related to Invasiveness in Non-Small Cell Lung Carcinoma Clin. Cancer Res., November 1, 2003; 9(14): 5282 - 5286. [Abstract] [Full Text] [PDF]
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E. A. Collisson, C. Kleer, M. Wu, A. De, S. S. Gambhir, S. D. Merajver, and M. S. Kolodney Atorvastatin prevents RhoC isoprenylation, invasion, and metastasis in human melanoma cells Mol. Cancer Ther., October 1, 2003; 2(10): 941 - 948. [Abstract] [Full Text]
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S. Tsutsumi, T. Taketani, K. Nishimura, X. Ge, T. Taki, K. Sugita, E. Ishii, R. Hanada, M. Ohki, H. Aburatani, et al. Two Distinct Gene Expression Signatures in Pediatric Acute Lymphoblastic Leukemia with MLL Rearrangements Cancer Res., August 15, 2003; 63(16): 4882 - 4887. [Abstract] [Full Text] [PDF]
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Q. Pan, L. W. Bao, and S. D. Merajver Tetrathiomolybdate Inhibits Angiogenesis and Metastasis Through Suppression of the NF{kappa}B Signaling Cascade Mol. Cancer Res., August 1, 2003; 1(10): 701 - 706. [Abstract] [Full Text] [PDF]
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T. Kamai, T. Tsujii, K. Arai, K. Takagi, H. Asami, Y. Ito, and H. Oshima Significant Association of Rho/ROCK Pathway with Invasion and Metastasis of Bladder Cancer Clin. Cancer Res., July 1, 2003; 9(7): 2632 - 2641. [Abstract] [Full Text] [PDF]
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 M. Cristofanilli, A. U. Buzdar, and G. N. Hortobagyi Update on the Management of Inflammatory Breast Cancer Oncologist, April 1, 2003; 8(2): 141 - 148. [Abstract] [Full Text] [PDF]
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 S. F. Doisneau-Sixou, P. Cestac, S. Chouini, J. S. Carroll, A. D. Hamilton, S. M. Sebti, M. Poirot, P. Balaguer, J.-C. Faye, R. L. Sutherland, et al. Contrasting Effects of Prenyltransferase Inhibitors on Estrogen-Dependent Cell Cycle Progression and Estrogen Receptor-Mediated Transcriptional Activity in MCF-7 Cells Endocrinology, March 1, 2003; 144(3): 989 - 998. [Abstract] [Full Text] [PDF]
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 R. M. Wolf, N. Draghi, X. Liang, C. Dai, L. Uhrbom, C. Eklof, B. Westermark, E. C. Holland, and M. D. Resh p190RhoGAP can act to inhibit PDGF-induced gliomas in mice: a putative tumor suppressor encoded on human Chromosome 19q13.3 Genes & Dev., February 15, 2003; 17(4): 476 - 487. [Abstract] [Full Text] [PDF]
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 S. A. Benitah, P. F. Valeron, H. Rui, and J. C. Lacal STAT5a Activation Mediates the Epithelial to Mesenchymal Transition Induced by Oncogenic RhoA. Mol. Biol. Cell, January 1, 2003; 14(1): 40 - 53. [Abstract] [Full Text]
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W. T. Arthur, S. M. Ellerbroek, C. J. Der, K. Burridge, and K. Wennerberg XPLN, a Guanine Nucleotide Exchange Factor for RhoA and RhoB, But Not RhoC J. Biol. Chem., November 1, 2002; 277(45): 42964 - 42972. [Abstract] [Full Text] [PDF]
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L. F. Su, Z. Wang, and M. J. Garabedian Regulation of GRIP1 and CBP Coactivator Activity by Rho GDI Modulates Estrogen Receptor Transcriptional Enhancement J. Biol. Chem., September 27, 2002; 277(40): 37037 - 37044. [Abstract] [Full Text] [PDF]
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 P. A. Solski, W. Helms, P. J. Keely, L. Su, and C. J. Der RhoA Biological Activity Is Dependent on Prenylation but Independent of Specific Isoprenoid Modification Cell Growth Differ., August 1, 2002; 13(8): 363 - 373. [Abstract] [Full Text] [PDF]
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K. L. van Golen, L. Bao, M. M. DiVito, Z. Wu, G. C. Prendergast, and S. D. Merajver Reversion of RhoC GTPase-induced Inflammatory Breast Cancer Phenotype by Treatment with a Farnesyl Transferase Inhibitor Mol. Cancer Ther., June 1, 2002; 1(8): 575 - 583. [Abstract] [Full Text] [PDF]
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 C. G. Kleer, K. L. van Golen, Y. Zhang, Z.-F. Wu, M. A. Rubin, and S. D. Merajver Characterization of RhoC Expression in Benign and Malignant Breast Disease : A Potential New Marker for Small Breast Carcinomas with Metastatic Ability Am. J. Pathol., February 1, 2002; 160(2): 579 - 584. [Abstract] [Full Text] [PDF]
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