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[Cancer Research 65, 6692-6700, August 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

ß1A Integrin Expression Is Required for Type 1 Insulin-Like Growth Factor Receptor Mitogenic and Transforming Activities and Localization to Focal Contacts

Hira Lal Goel1, Michael Breen1, Jianzhong Zhang1, Ishita Das1, Sadie Aznavoorian-Cheshire1, Norman M. Greenberg2, Ada Elgavish3 and Lucia R. Languino1

1 Department of Cancer Biology and the Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts; 2 Cancer Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington; and 3 Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama

Requests for reprints: Lucia R. Languino, Department of Cancer Biology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605. Phone: 508-856-1606; Fax: 508-856-3845; E-mail: lucia.languino{at}umassmed.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cells' ability to proliferate in response to growth factor stimulation is significantly altered during cancer progression. To investigate the mechanisms underlying these alterations in prostate cancer, the role and expression of ß1A integrin and type 1 insulin-like growth factor receptor (IGF-IR), known to contribute to cell proliferation and transformation, were analyzed. Using small interfering RNA oligonucleotides to down-regulate ß1A, we show that ß1A expression is required for IGF-IR–mediated prostate cancer cell proliferation and anchorage-independent growth. In vivo, using age-matched transgenic adenocarcinoma of mouse prostate (TRAMP) mice at different stages of prostate cancer [prostatic intraepithelial neoplasia, PIN; well-differentiated adenocarcinoma, WD; and poorly differentiated adenocarcinoma, PD], the expression of ß1A and of IGF-IR was studied. ß1A and IGF-IR expression levels were concurrently up-regulated in high PIN and WD, whereas their expression did not correlate in late-stage PD. In contrast to the up-regulated expression of ß1A, the levels of ß1C, a ß1 cytoplasmic variant that inhibits cell proliferation, were down-regulated in all stages of prostate cancer. A similar expression pattern was observed for a ß1C downstream effector, Grb2-associated binder-1 (Gab1) which is known to inhibit IGF-IR phosphorylation. To analyze in vitro the mechanistic implications of ß1A, ß1C, and Gab1 deregulation in prostate cancer, we investigated whether expression of either ß1 variant in ß1-null cells affected IGF-IR localization. We found that IGF-IR and ß1A were colocalized in highly specialized integrin signaling compartments, designated focal contacts. However, in the presence of ß1C, IGF-IR remained diffuse on the cell surface and did not localize to focal contacts. The findings that ß1 integrins and IGF-IR are concurrently deregulated and that expression of ß1 integrins is necessary to achieve appropriate IGF-IR intracellular distribution point to the important role that the cross-talk between these receptors may have during prostate cancer progression and will be helpful in formulating new therapeutic strategies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate cancer remains a significant health issue for aging men and is the second leading cause of cancer death among American men (1). Whereas substantial advances have been made in the diagnosis and treatment of prostate cancer, the underlying causes of this disease remain to be fully investigated. The development of prostate cancer proceeds through a series of defined stages, including preinvasive disease (prostatic intraepithelial neoplasia, PIN), invasive cancer, and androgen-dependent or androgen-independent metastases (2, 3).

The ability of neoplastic prostate cells to grow and invade is mediated by interactions with the surrounding cells and extracellular matrix–mediated by integrins (4). Integrins are crucial regulators of differentiation, growth, survival, migration, and invasion (5, 6). In prostate cancer, tumor cells have a markedly different surrounding matrix than normal cells; thus, changes in the integrin profile may be functionally relevant and contribute to cancer progression (7, 8). A number of studies have reported changes in integrin expression as prostate cancer progresses to an advanced stage (7, 9, 10).

Integrins are heterodimers consisting of an {alpha} and ß subunit (6). The cytoplasmic domain of the ß1 subunit, in its canonical form (ß1A), is highly conserved and is a positive regulator of cell proliferation, development, migration, and integrin localization (11, 12). The ß1C integrin is an alternatively spliced variant of the ß1 subfamily that contains a unique sequence in its cytoplasmic domain (12). Previous studies have shown that either full-length ß1C or its cytoplasmic domain inhibits prostate cancer cell proliferation (12). ß1C is expressed in the nonproliferative, differentiated epithelium and is selectively down-regulated in prostatic adenocarcinoma (13–16).

Several reports in the literature suggest that there are physical and functional interactions between integrins and components of growth factor signaling pathways, including insulin-like growth factor (IGF) or its downstream signaling proteins (17). However, the role of these interactions between type 1 IGF receptor (IGF-IR) and integrins in regulating the progression of prostate cancer is still not clear. The IGF axis is an important modulator of growth and development, and changes in this axis may have important implications in malignant growth (18, 19). The IGF and its receptor (IGF-IR) are found in human prostate stroma and epithelial cells (20). There is, however, no consensus regarding the relative levels of IGF-IR expression in benign and malignant prostate epithelium and the role of IGFs in metastases. In 1996, Tennant et al. reported that IGF-IR expression is significantly lowered at the protein and RNA level in malignant versus benign prostate epithelium (21). Similarly, Chott et al. reported that IGF-IR is expressed in luminal and basal epithelium in nonneoplastic prostate but is lost in metastatic androgen-independent prostate cancer (22). However, Kaplan et al. did fail to show a significant increase in IGF-IR mRNA during primary prostate tumor progression in transgenic adenocarcinoma of mouse prostate (TRAMP) mice, indicating the possibility of posttranscriptional regulation of IGF-IR but showed reduced levels of IGF-IR mRNA in metastatic lesions and in androgen-independent disease (23). Subsequently, Nickerson et al. reported that progression to androgen independence is associated with increased expression of IGF-I and IGF-IR as well as increased tyrosine phosphorylation of IGF-IR (24). Finally, in 2002, another study has shown that IGF-IR was significantly up-regulated at the protein and mRNA level in primary prostate cancer compared with benign prostatic epithelium (25).

Most signaling pathways stimulated by receptor tyrosine kinases use scaffolding adaptors like insulin receptor substrate (IRS) or Gab known to be involved in IGF signaling axis (26, 27). Upon the addition of growth factors, Grb2-associated binder (Gab1) becomes tyrosine phosphorylated and recruits Src homology-2 domain-containing tyrosine phosphatase 2 (Shp2; ref. 28). In our previous study, we have shown that the Gab1/Shp2 complex is recruited to the cell surface by integrins. Upon recruitment to cell surface, Gab1/Shp2 complex causes IGF-IR dephosphorylation and causes change in cell adhesion to laminin in response to IGF-I (29). Previous studies have shown that overexpression of Gab1 increases growth factor responsiveness and tumorigenicity in NIH3T3 cells (26). A recent study has shown that overexpression of Gab1 lacking the pleckstrin homology domain enhances (by 3-fold), whereas wild-type Gab1 inhibits, epidermal growth factor–induced soft agar colony formation of preneoplastic Syrian hamster cells (30). However, studies carried out to investigate whether changes in expression levels of Gab1 occur during cancer progression have not been done.

In the present study, we show that IGF-IR–mediated prostate cancer cell proliferation and anchorage-independent growth is inhibited by ß1A down-regulation. Our study highlights a role for the ß1A integrin as modulator of IGF-IR–mediated functions in prostate cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies. The following mouse monoclonal antibodies (mAb) were used: to all human or mouse ß1 integrins, Clone-18 (BD Bioscience, San Jose, CA); to human ß1 integrin (K-20; Immunotech, Marseille, France); to phosphatidylinositol 3-kinase (PI3-K; p85; BD Bioscience); mouse IgM specific to ß1C integrin, 7D5/BF10 (31); to vinculin (Chemicon, Temecula, CA). The following rabbit polyclonal antibodies were used: to all ß1 integrins (M-106; Santa Cruz Biotechnology, Santa Cruz, CA); to Akt (Cell Signaling, Beverly, MA); to extracellular signal-regulated kinase-2 (ERK-2, Santa Cruz Biotechnology); to cytoplasmic domain of ß1A integrin (kindly provided by Dr. E. Ruoslahti, Cancer Research Center, The Burnham Institute, La Jolla, CA); antibody to IGF-IR– (Santa Cruz Biotechnology); to ß5 integrin (Chemicon); to ß1C integrin (13); to Gab1 (Upstate Biotechnology, Lake Placid, NY). Normal mouse IgM (ni-Ab) and normal rabbit IgG (rIgG) were from Calbiochem (La Jolla, CA).

Transgenic mice. TRAMP mice on a pure C57BL/6 background were bred in the colony at the University of Alabama at Birmingham School of Medicine. Transgenic females were bred with nontransgenic littermates (NT) males because transgenic males tend to develop prostate tumors. All mice were maintained in a climate-controlled environment with a 12-hour light/12-hour dark cycle and diet and water supplied ad libitum. Breeders were fed standard pellet mouse feed (Harlan Teklad 7012, Madison, WI). Following weaning at 3 to 4 weeks of age, the gender of offspring was determined, males were separated from females, and a tail biopsy was collected from each mouse. Tail DNA, isolated by standard procedures, was used for determination of transgene incorporation by PCR as described previously (32–34). All TRAMP mice develop premalignant changes resembling human PIN, which ultimately progress to poorly differentiated (PD) prostatic adenocarcinomas that metastasize to distant sites, primarily the lymph nodes, bones, and lungs (33). The present study focused on PD (grade 6) as an example of advanced prostate cancer, WD (grade 4) as an example of an early-stage of adenocarcinoma, prostates grade 3 as an example of PIN, and normal prostate (grade 1) from age-matched NT littermates.

Animal care and treatments were conducted in accordance with established guidelines and protocols approved by the University of Alabama Animal Care Committee.

Preparation and analysis of tissues. At necropsy, the urogenital tract, consisting of the prostate, the seminal vesicles, and the emptied bladder, was weighed. A fragment of the DLP, as well as periaortic lymph nodes, were rapidly frozen in liquid nitrogen and stored at –80°C, until further processed. The rest of the DLP was fixed in an acid alcohol solution containing 96% ethanol, 1% glacial acetic acid, and 3% distilled water, as described (35). Lymph nodes were fixed in 10% neutral buffered formalin phosphate. The fixed tissues were then embedded in paraffin and 4- to 5-µm sections were mounted on Colorfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Sections were stained with H&E (lymph nodes) or with Gomori trichrome staining (DLP). DLP sections were evaluated blindly and scored using established criteria (32, 34) as normal prostate (score 1), low PIN (score 2), high PIN (score 3), well-differentiated (WD, score 4), moderately differentiated (MD, score 5), or poorly differentiated (PD, score 6). Prostate sections from each mouse were evaluated blindly by Dr. Isam A. Eltoum, a certified Pathologist (Department of Pathology, University of Alabama at Birmingham, Birmingham, Al; refs. 32, 34). Tumors showing grade 2 to 5 were heterogeneous and the pathologist graded them depending on the type of tissue that is most prevalent. The MMHCC Pathology Committee has published very elaborate guidelines in Cancer Research (36). Dr. Isam A. Eltoum, the pathologist who has examined these sections has followed those guidelines during his assessments of the tumor grade.

Data analysis. Analysis of the expression levels of IGF-IR and ß1 integrin was done using SigmaStat, version 3.0 (SysStat, Inc., Evanston, IL). When the effect of one variable was examined, one-way ANOVA was done if data in multiple groups were normally distributed with equal variance. The Kruskall-Wallis one-way ANOVA on ranks was used if three or more groups were compared and the data were not normally distributed with equal variance. If the latter two tests indicated that the groups were significantly different, a Tukey or Dunn test was carried out, respectively, to determine which of the groups were significantly different from the others. P values for the appropriate tests are given with the results.

Prostate tissue lysis. TRAMP mice were maintained as described above until they were 28 weeks of age. DLPs were collected at necropsy. In this study, we used frozen prostate samples from TRAMP graded 3, 4, and 6 as well as prostate from age-matched NT. Tumor specimens were extracted as follows: specimens were homogenized on ice in a Dounce homogenizer in 100 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.1% Triton X-100, 1 mmol/L benzamidine, 10 µg/mL soybean trypsin inhibitor, 10 µg/mL leupeptin, 1 mmo/L phenylmethylsulfonyl fluoride, 1 µg/mL pepstatin A, 1 µmol/L calpain inhibitor, 1 mmol/L Na3VO4, and 1 mmol/L Na4O7P2. SDS was then added to a final concentration of 5%, after which the extracts were boiled for 5 minutes. After boiling, samples were centrifuged at 14,000 x g for 20 minutes, and supernatant was collected for protein determination using bicinchoninic acid protein assay reagent (Pierce, Rockford, IL).

Immunoblot analysis. Proteins from TRAMP prostate tissue lysate (PIN, WD, and PD) or from NT were electrophoresed using 7.5% SDS-PAGE under reducing condition and transferred onto polyvinylidene difluoride membranes, then probed sequentially with mAb to ß1 integrins (Clone-18) or to PI3-K or to IGF-IR-ß, mouse IgM to ß1C integrin (7D5/BF10; ref. 31), normal mouse IgM, rabbit polyclonal antibody to ß1C integrin or Gab1, or to ß5 integrin. Proteins were detected using enhanced chemiluminescence.

Immunofluorescence microscopy. GD25 murine cells transiently transfected with human ß1A or ß1C integrin or GD25 cells stably transfected with human ß1A integrin using LipofectAMINE 2000 (Invitrogen, San Diego, CA) were cultured as described previously (37). Cells were seeded onto 10 µg/mL fibronectin-coated coverslips and processed for immunofluorescence microscopy as described previously (38). The primary antibodies used were K-20 to human ß1 integrin or a mAb to vinculin or a polyclonal antibody to IGF-IR-ß. The secondary antibodies used were goat anti-rabbit IgG coupled to FITC or goat anti-mouse IgG coupled to rhodamine (The Jackson Laboratory, West Grove, PA). One hundred cells per experiment to study the colocalization of IGF-IR and ß1 integrins were examined and experiments were done >10 times.

Small interfering RNA–mediated down-regulation of ß1A integrin. This study used small interfering RNA (siRNA) to target a region specific to the ß1A transcript. The siRNA was designed using conventional criteria (39) and corresponded to nucleotides 2425 to 2443 of the coding region in human ß1A integrin. The sequence of siRNA was sense strand 5'-AUGGGACACGGGUGAAAAUTT-3' and antisense strand 5'-AUUUUCACCCGUGUCCCAUTT-3'. These sequences were submitted to BLAST search to ensure that only the ß1A integrin was targeted by the ß1A integrin siRNA and that control sequences were not homologous to any known genes. siRNA specific to ß1A integrin or control were synthesized by Dharmacon (Lafayette, CO). Mouse ß1A integrin also have the same last 18 nucleotides in its coding regions as the human ß1A integrin and correspond to nucleotides 2502 to 2519 of mouse ß1A integrin coding region.

PC3, R–, or R+ cells were cultured as described before (29). TRAMP-C2 (American Type Culture Collection, Manassas, VA) cells were grown in DMEM, 2 mmol/L L-glutamine, 5 mg/mL bovine insulin, 10 nmol/L dihydrotestosterone, 5% fetal bovine serum (FBS), and 5% Nu-Serum (BD Biosciences, Bedford, MA). TRAMP-C2, R+, R–, or PC3 cells were transfected with siRNA (100 nmol/L) using oligofectamine (Invitrogen) in serum-free medium. After a 4-hour incubation, cells were fed with complete growth medium described above and incubated for additional 48 hours. After incubation, cells were lysed and proteins were separated on SDS-PAGE under reducing conditions. Proteins were immunoblotted with a mAb to ß1 integrins (Clone-18) or to Akt or to ERK.

Sulforhodamine B assay. Proliferation was measured using sulforhodamine B (SRB) assay. PC3, R+, or TRAMP-C2 cells were transfected with siRNA to ß1A integrin or control siRNA or oligofectamine alone for 48 hours. Cells were detached and seeded on 96-well cell culture plate (7.5 x 103 cells per well) and allowed to attach for 5 hours in the presence of serum. After attachment, cells were washed and incubated in the presence or in the absence of IGF-I (100 ng/mL) in serum-free medium for 48 hours. After incubation, cells were fixed and stained with SRB as described before (29). As a plating control, one set of plates after 5 hours of cell attachments was washed, fixed, and stained with SRB.

Anchorage-independent growth assay. Cell growth in soft agar was assayed by scoring the number of colonies formed in medium (DMEM supplemented with 10% FBS) containing 0.3% agarose, with a 0.5% agarose medium underlay as described (40). R– or R+ (2 x 103) or DU145 (5 x 103) cells were seeded on 60-mm diameter plates in triplicate in the presence of IGF-I (100 ng/mL). Cells were fed with 1.5 mL of suspension medium (DMEM supplemented with 10% FBS) every 3 days. The number of colonies larger than 100 µm were counted after 14 days (R+ cells) or after 21 days (DU145 cells). Cell viability was measured 48 hours after siRNA transfection using trypan blue exclusion and plotted as percentage of total viable cells.

Immunohistochemistry. Paraffin-embedded prostate tissue sections from eight mice were baked for 2 hours at 60°C and washed thrice with xylene to deparaffinize the slides. The sections were rehydrated using ethanol and then distilled water. The sections were boiled in sodium citrate using a steamer at 100°C to enhance antigen retrieval. Endogenous peroxidase was quenched with 3% H2O2 for 5 minutes. Blocking was achieved with 50% normal goat serum in TBS containing 0.2% bovine serum albumin for 20 minutes at room temperature. The sections were then incubated with primary antibodies (rabbit polyclonal antibody to ß1 integrin, M-106, 0.8 µg/mL or ni-rIgG, 0.8 µg/mL) overnight at 4°C, followed by biotinylated goat anti-rabbit IgG (3 µg/mL, Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature. The sections were incubated with streptavidin-peroxidase complex (5 µg/mL, Vector Laboratories) for 30 minutes at room temperature. Each incubation step was followed by three washes with TBS. Finally, 3,3'-diaminobenzidine tetrahydrochloride dehydrate was used as chromogen (Sigma, St. Louis, MO). Sections were then stained using hematoxylin for 1 minute. Tissue sections were examined on an Olympus BX41 microscope and photographed using Olympus DP12 camera. The immunostaining of ß1A integrin was independently evaluated by four investigators, including Dr. David S. Garlick (DVM, Pathologist, Department of Cancer Biology, University of Massachusetts Medical School), H.L.G., M.B., and L.R.L. The expression of ß1 integrin in prostate tissue sections of NT or TRAMP was evaluated and recorded as a separate observation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ß1A expression is essential for insulin-like growth factor-I–mediated prostate cancer cell proliferation. To investigate the role of ß1A in IGF-I stimulated prostate cancer cell proliferation, we down-regulated the expression of ß1A in PC3 or TRAMP-C2 cells using siRNA specific to ß1A integrin. As shown in Fig. 1A and B, transfection of ß1A siRNA significantly down-regulated ß1A expression compared with control siRNA, without affecting the expression of other molecules such as Akt (Fig. 1A), ERK (Fig. 1B), or cell viability (data not shown). To investigate the role of ß1A in IGF-I stimulated cell proliferation, we transfected PC3 (Fig. 1C) or TRAMP-C2 (Fig. 1D) cells with ß1A siRNA or control siRNA and measured their proliferative response in the presence or in the absence of IGF-I. Cells transfected with ß1A siRNA did not respond to IGF-I (Fig. 1C and D, left), whereas IGF-I increased cell proliferation when the cells were transfected with control siRNA (Fig. 1C and D, left). Cells transfected with control siRNA or ß1A siRNA did attach to tissue culture plates in a comparable manner; therefore, the effect observed was not a consequence of a differential ability of these cells to adhere to the plate (Fig. 1C and D, right). Based on these results, we conclude that ß1A integrin expression is essential for IGF-I-mediated prostate cancer cell proliferation.



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  		Figure 1. ß1A expression is essential for IGF-I-mediated prostate cancer cell proliferation. PC3 (A and C) and TRAMP-C2 (B and D) cells were transfected with 100 nmol/L ß1A integrin siRNA, control siRNA or oligofectamine alone (OLF). A-B, after 48 hours, cells were detached, lysed, and proteins were separated using SDS-PAGE and immunoblotted with mAb to ß1 integrins (Clone-18) or antibody to Akt or antibody to ERK. C-D, cells were plated in 96-well tissue culture plates in the presence of 10% FBS for 5 hours. Cells were washed, incubated for 48 hours in serum-free medium in the presence or in the absence of IGF-I (100 ng/mL), fixed, and stained with SRB (left). As plating control, one plate was fixed and stained with SRB after 5 hours in the presence of serum (right). In (C) and (D), the differences in cell proliferation in the presence or in the absence of IGF-I are statistically significant. *, P ≤ 0.01.

 
ß1A is required for type 1 insulin-like growth factor receptor–mediated cell anchorage-independent growth. IGF-I binds to its receptor, IGF-IR, and stimulates tumor cell growth and cell survival (18). Cells devoid of IGF-IR are unable to undergo transformation, suggesting a crucial role for IGF-IR in the establishment and maintenance of the transformed phenotype (41). To investigate the role of ß1A in IGF-IR-mediated prostate cancer cell transformation, we used R– or R+ cells. R– cells are 3T3-like fibroblasts established from mouse embryos with a targeted disruption of igf-ir gene and cannot be transformed by SV40 T antigen, or an activated Ha-ras, or a combination of both (42–44). R+ cells were generated by transfecting wild-type IGF-IR in R– cells (42–44). We down-regulated ß1A in R+ cells using siRNA specific to ß1A integrin and measured the effect of IGF-I on cell proliferation. We found significant down-regulation of ß1A integrin upon siRNA transfection in R+ cells (Fig. 2A). As shown in Fig. 2B, ß1A down-regulation prevented IGF-I stimulated R+ cell proliferation (Fig. 2B, left), but it did not affect cell attachment (Fig. 2B, right). In R– cells, we observed significant cell death (90%) after transfection with ß1A siRNA as compared with R+ cells, indicating a crucial role for ß1A integrin in cell survival in the absence of IGF-IR (Fig. 2C). An analysis of the effect of ß1A integrin down-regulation on the cells' ability to grow in anchorage-independent manner shows that R+ cells formed colonies in soft agar when transfected with control siRNA, but their ability to grow in soft agar was completely inhibited by ß1A down-regulation (Fig. 2D). R– cells did not form colonies in soft agar (Fig. 2D) confirming a previous published observation that IGF-IR is required for anchorage-independent growth (40). Similar results were obtained in human prostate cancer cells, DU145, whose viability was not affected by ß1A integrin down-regulation, although a complete inhibition of anchorage-independent growth was achieved (data not shown). These results show that ß1A integrin expression plays a crucial role in IGF-IR-mediated cell transformation.



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  		Figure 2. ß1A is required for IGF-IR mediated anchorage-independent growth. A-B, R+ cells were transfected with 100 nmol/L ß1A integrin siRNA, control siRNA or oligofectamine alone (OLF). A, after 48 hours, cells were detached, lysed and proteins were separated using SDS-PAGE and immunoblotted with a mAb to ß1 integrins (Clone-18) or antibody to Akt. B, cells were plated on 96-well tissue culture plates in the presence of 10% FBS for 5 hours. Cells were washed, incubated for 48 hours in serum-free medium in the presence or in the absence of IGF-I (100 ng/mL), fixed, and stained with SRB (left). As plating control, one plate was fixed and stained with SRB after 5 hours in the presence of 10% FBS (right). In (B), the differences in cell proliferation in the presence or in the absence of IGF-I are statistically significant. *, P ≤ 0.01. C, R– or R+ cell viability after transfection with control siRNA or ß1A integrin siRNA was measured. The number of viable cells and of total cells was counted and the percentages of viable cells were calculated. D, R– or R+ (2 x 103) cells were suspended in 1.5-mL medium containing 0.3% agarose and 10% FBS, with a 0.5% agarose medium underlay in 60-mm diameter plates in the presence of IGF-I (100 ng/mL). Cells were fed with 1.5-mL suspension medium (DMEM supplemented with 10% FBS) every 3 days. The number of colonies larger than 100 µm were counted after 14 days. Triplicate observations in three separate experiments were done. Data from one representative experiment.

 
ß1A and type 1 insulin-like growth factor receptor expression is up-regulated in prostatic intraepithelial neoplasia and well-differentiated prostate carcinoma. Because we have shown that the ß1A integrin regulates IGF-IR-mediated transformation (29), we investigated whether the expression levels of ß1A and IGF-IR correlate during prostate cancer progression in vivo. We studied the expression profile of the ß1A integrin and IGF-IR in prostate tissues from TRAMP mice or from NT. We have analyzed five prostates showing tumor grade 3, five prostates showing tumor grade 4, and four prostates showing tumor grade 6. As shown in Fig. 3A-B, we found a significant increased expression of IGF-IR and ß1A integrin in all prostate tissues showing grades 3 and 4 compared with prostates from NT mice (P < 0.011). The concurrent up-regulation of ß1A and IGF-IR in the early stages of neoplastic transformation in TRAMP mice is consistent with our model that ß1A and IGF-IR form a functional complex. In PD tumors, however, the expression of ß1A was significantly decreased relative to WD tissues, whereas levels of IGF-IR were comparable with WD tissues, suggesting that enhanced expression of ß1A is not required to maintain the proliferative capacity of late-stage tumor cells (Fig. 3C). Immunohistochemical analysis of TRAMP prostate tissue sections was done in a qualitative manner to determine the localization of ß1 integrin. As shown in Fig. 4, we consistently found that the ß1 integrin was expressed in epithelial and stroma cells in WD but was down-regulated in PD tissue specimens. These results indicate that a concurrent expression of IGF-IR and ß1 occurs in PIN and WD but not in PD prostate cancer in TRAMP mice.



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  		Figure 3. ß1A integrin and IGF-IR are upregulated in high-grade PIN and WD TRAMP prostatic adenocarcinoma. Prostate tumor lysates from 28-week-old TRAMP mice and their corresponding NT were separated using SDS-PAGE and proteins were immunoblotted with mAb to ß1 integrins (Clone-18), rabbit polyclonal antibody to ß1A integrin, to IGF-IR or, as loading control, mAb to PI3-K (p85). A-B, NT, grade 3 (PIN), grade 4 (WD). C, grade 6 (PD). The results are presented as fold increase obtained using densitometric analysis upon normalization. Two sets of prostate tissues in two separate gels.

 


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  		Figure 4. ß1 integrin is down-regulated in PD TRAMP prostatic adenocarcinoma. Paraffin sections (4-5 µm) of DLP from 28-week-old TRAMP mice were stained with H&E (A and D) antibodies against ß1 integrin (B and E) or ni-rIgG (C and F). A-C, grade 4 (WD); D-F, grade 6 (PD). Magnification, 20x.

 
Down-regulation of ß1C integrin and Gab1 expression in prostate carcinoma. The ß1C integrin is expressed in human nonproliferative, differentiated epithelium and is selectively down-regulated in prostatic adenocarcinoma (13–16). Similarly, we show here using the TRAMP model of progressive prostate carcinoma that ß1C is expressed in normal mouse prostate (Fig. 5A), but its expression is down-regulated in PIN, WD, and PD adenocarcinomas (Fig. 5B). Detergent lysates of PNT1A cells were used as a positive control (Fig. 5B, lane 1). In contrast, ß5 integrin expression was found in normal (NT) and TRAMP prostates at similar levels (Fig. 5B). These data indicate that selective down-regulation of ß1C is an early event during prostate cancer progression.



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  		Figure 5. Down-regulation of ß1C integrin and Gab1 in PIN, WD, and PD TRAMP prostate tissues. A, PNT1A cell lysate (lane 1) or tissue extract from normal mouse prostate (lane 2) were separated using a 7.5% SDS-PAGE and proteins were immunoblotted with mAb to ß1C integrin or nonimmune antibody (ni-Ab). B, PNT1A cell lysate or prostate tumor lysates from 28-week-old TRAMP mice and their corresponding NT were separated using SDS-PAGE and proteins were immunoblotted with rabbit polyclonal antibody to ß1C integrin or ß5 integrin. C, PNT1A cell lysate or prostate tumor lysates from 28-week-old TRAMP mice and their corresponding NT were separated using SDS-PAGE and proteins were immunoblotted with rabbit polyclonal antibody to Gab1 or ß5 integrin.

 
In our previous study, we have shown that ß1C recruits Gab1 to the cell surface and that Gab1 mediates ß1C-negative regulation of IGF-IR signaling (29). A potential correlation of the expression of ß1C and Gab1 was investigated. We analyzed Gab1 expression in tissue lysates from NT, and from PIN, WD and PD TRAMP prostates using an antibody to Gab1. Consistent with the expression profile of ß1C integrin, Gab1 seems down-regulated in all TRAMP prostate specimens of grades 3, 4, and 6 compared with normal (NT) prostate tissue specimens (Fig. 5C). These results indicate that expression of ß1C integrin and Gab1 correlates in both normal and neoplastic prostate tissues.

ß1A integrin recruits type 1 insulin-like growth factor receptor to focal contacts. In our previous study, we have shown that ß1A but not ß1C associates with IGF-IR and that ß1C expression down-regulates IGF-IR-mediated signaling (29). To investigate the mechanism through which ß1 integrin regulates IGF-IR signaling, we hypothesized that ß1 might regulate the localization of IGF-IR in specific cellular signaling compartments. The experiments were done using GD25 cells that do not express ß1 integrin, stably or transiently transfected with ß1A or ß1C. In ß1A stable transfectants, we found that IGF-IR was localized in focal contacts, where vinculin (Fig. 6A-C), ß1A and several signaling molecules, such as Src and focal adhesion kinase (45), are known to be localized in spread cells. In transiently transfected cells, ß1A and IGF-IR colocalized in focal contacts (Fig. 6D-F), whereas upon ß1C expression, IGF-IR was not found to be localized in focal contacts (Fig. 6G-I, arrow). Moreover, in nontransfected GD25 cells, the IGF-IR was found to be diffuse and not localized to focal contacts, suggesting that ß1A integrin is essential for the localization of IGF-IR to focal contacts (Fig. 6G-I, arrowhead). In conclusion, these data indicate that IGF-IR localization is controlled by specific ß1 integrins.



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  		Figure 6. ß1A integrin but not ß1C recruits IGF-IR to focal contacts. A-C, GD25 cells stably transfected with human ß1A integrin were stained with antibodies to IGF-IR (green) or vinculin (red); image-merging analysis of vinculin and IGF-IR (yellow). D-I, GD25 cells transiently transfected with human ß1A (D-F) or ß1C (G-I, arrow) integrin were stained with antibodies to IGF-IR (green) or to ß1 integrins (red); image-merging analysis of ß1 integrins and IGF-IR (yellow). G-I, nontransfected GD25 cell (arrowhead). The experiments were repeated 10 times with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The novel observation of this study is that down-regulation of the ß1A integrin via siRNA oligonucleotides results in inhibition of IGF-IR-mediated cell proliferation and anchorage-independent growth. It is also shown that whereas ß1A and IGF-IR are up-regulated, the ß1C integrin cytoplasmic variant and Gab1, a ß1C downstream effector, are down-regulated during prostate cancer progression. Finally, ß1A's ability to modulate IGF-IR localization in focal contact signaling compartments, is shown to be the mechanism through which ß1A controls IGF-IR signaling.

This is the first study that uses siRNA oligonucleotides to integrins to prove a role for the integrin/growth factor receptor cross-talk in prostate cancer and suggests the use of siRNA to down-regulate integrins as a novel therapeutic approach for this disease. The results show that IGF-mitogenic and transforming activities but neither basal proliferation nor cell viability are prevented by siRNA to ß1A integrin. Based on this observation, we conclude that ß1A expression is essential to activate IGF-IR.

It is well established that activated IGF-IR sends signals required for growth and transformation (41). Our results indicate that the synergistic action of IGF-IR and ß1A in stimulating mitogenic signals during initiation of carcinogenesis is required. In conclusion, a model can be formulated in which differential expression and function of ß1 integrins in prostate cancer modulates cell responses to IGF via IGF-IR. We suggest a paradigm shift that changes the point of view that (a) ß1 integrins per se contribute to tumor progression (46, 47) and that (b) the IGF-IR per se contributes to tumor progression (18). By elucidating the players that contribute to prostate cancer progression, future studies will provide new insights into the current therapeutic approaches that target either integrins or IGF-IR.

The molecular basis and the cross-talk between the ß1 integrins and IGF-IR in prostate cancer progression have not been investigated. Our previous study has unraveled a novel mechanism mediated by ß1 integrin cytoplasmic variants that differentially regulates IGF-mediated prostate cell adhesion to a basement membrane protein, laminin (29). The relevance of this observation to prostate cancer, where up-regulation of the IGF-IR has been described (24), is that ß1A by limiting cancer cell adhesion to basement membrane proteins and promoting cell proliferation in response to IGF presumably allows the tumor mass to expand and invade. In contrast, ß1C, a cytoplasmic variant known to be down-regulated in prostate cancer, has the opposite effect; it completely prevents IGF-mediated cell proliferation and tumor growth, and promotes cell adhesion to laminin. Interestingly, the loss of IGF-IR in the adult prostate seems to cause the cells to become greatly disorganized and loose attachment to basement membrane while remaining viable.4 Consistent with the hypothesis that ß1A integrin and IGF-IR act as a complex that promotes cell proliferation but inhibits cell adhesion to basement membrane, in this study, ß1A and IGF-IR are found to be concurrently up-regulated in TRAMP prostate tissues in the early stages of neoplastic transformation, when disruption of the basement membrane occurs.

We describe a concurrent increase in expression of both ß1A integrin and IGF-IR in PIN and WD tumors in a defined model of prostate cancer, designated TRAMP; in contrast, PD tumors, where ß1A is down-regulated, no longer show this correlation. As previously described for human prostate cancer (7), the present study shows that the expression of ß1 (ß1A and ß1C) is altered during cancer progression in TRAMP mice. The findings show, specifically, that ß1A integrin expression is up-regulated, whereas ß1C is down-regulated during prostate tumor progression. This suggests that up-regulated expression of ß1A and IGF-IR might contribute to uncontrolled cell division during initiation of prostate cancer; however, increased levels of both molecules are not required in the late stages of tumor progression. It is, thus, expected that lack of expression of ß1A will adversely affect initiation of prostate cancer growth. This is likely to be the result because, similarly, targeted disruption of ß1 has been recently shown to prevent initiation of mammary tumorigenesis in vivo in a transgenic mouse model (47).

The mechanism by which the ß1 integrin cytodomain differentially modulates prostate cancer cell functions in response to IGF and allows IGF-IR to dissect downstream signaling pathways occurs via differential recruitment of IGF-IR downstream effectors to the plasma membrane. A specific association between integrins and IGF-IR effector molecules, specifically ß1A and IRS-1, ß3 integrins and IRS-1, ß3 integrins and DOK1, and ß1C and Gab1/Shp2-phosphatase complex, is likely to explain many activities attributed to IGF-IR (29, 48–50). In this study, we show that both ß1 integrins and IGF-IR are found to be colocalized in focal contacts where a significant amount of integrin downstream signaling is coordinated. In contrast, expression of ß1C that is known to remain diffuse on the cell surface, prevents, or is not able to support IGF-IR localization in focal contacts. The relevance of these findings to prostate cancer is high because both ß1C and Gab1 are down-regulated in prostate cancer when ß1A is up-regulated. Presumably, in normal tissues, by preventing IGF-IR localization to focal contacts, ß1C potentiates Gab1/Shp2 signaling that in turn keeps IGF-IR in a nonphosphorylated state. In contrast, in neoplastic tissues, an appropriate localization of IGF-IR in focal contacts mediated by ß1A association with IGF-IR maintains IRS-1 signaling active and supports tumor progression.

In conclusion, deregulated expression of integrins and of IGF-IR and deregulation of their cross-talk is likely to be responsible for several alterations of cell functions and intracellular signaling in prostate cancer. Therefore, these alterations should be regarded as highly significant events in the search for novel therapeutic targets in prostate cancer.


   Acknowledgments
 
Grant support: NIH grants RO1 CA-89720 and CA109874 (L.R. Languino) and RO1 CA-82807 (N.M. Greenberg), Army grants PCRP DAMD17-98-1-8506 and PCRP DAMD PC040221 (L.R. Languino), and American Institute for Cancer Research grant AICR 00A023 (A. Elgavish).

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.

We thank Natalia Teider for technical assistance with the immunofluorescence studies, Drs. David S. Garlick and Isam A. Eltoum for analyses of immunohistochemical staining, and Marjory Thomas for helping with the article preparation.


   Footnotes
 
4 N.M. Greenberg, unpublished results. Back

Received 12/ 3/04. Revised 3/24/05. Accepted 5/13/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Tiwari RC, Murray T, et al. Cancer statistics, 2004. CA Cancer J Clin 2004;54:8–29.[Abstract/Free Full Text]
  2. Nicholson B, Theodorescu D. Molecular therapeutics in prostate cancer. Histol Histopathol 2003;18:275–98.[Medline]
  3. Scher HI, Heller G. Clinical states in prostate cancer: toward a dynamic model of disease progression. Urology 2000;55:323–7.[CrossRef][Medline]
  4. Boudreau N, Myers C, Bissell MJ. From laminin to lamin: regulation of tissue-specific gene expression by the ECM. Trends Cell Biol 1995;5:1–4.[CrossRef][Medline]
  5. Quaranta V, Plopper GE. Integrins and laminins in tissue remodeling. Kidney Int 1997;51:1441–6.[Medline]
  6. Ruoslahti E. Fibronectin and its integrin receptors in cancer. Adv Cancer Res 1999;76:1–20.[Medline]
  7. Fornaro M, Manes T, Languino LR. Integrins and prostate cancer metastases. Cancer Metastasis Rev 2003;20:321–31.
  8. Bogenrieder T, Herlyn M. Axis of evil: molecular mechanisms of cancer metastasis. Oncogene 2003;22:6524–36.[CrossRef][Medline]
  9. Murant SJ, Handley J, Stower M, Reid N, Cussenot O, Maitland NJ. Coordinated changes in expression of cell adhesion molecules in prostate cancer. Eur J Cancer 1997;33:263–71.
  10. Knox JD, Cress AE, Clark V, et al. Differential expression of extracellular matrix molecules and the {alpha}6-integrins in the normal and neoplastic prostate. Am J Pathol 1994;145:167–74.[Abstract]
  11. Hemler ME. Integrin associated proteins. Curr Opin Cell Biol 1998;10:578–85.[CrossRef][Medline]
  12. Fornaro M, Languino LR. Alternatively spliced variants: a new view of the integrin cytoplasmic domain. Matrix Biol 1997;16:185–93.[CrossRef][Medline]
  13. Fornaro M, Tallini G, Bofetiado CJM, Bosari S, Languino LR. Down-regulation of ß1C integrin, an inhibitor of cell proliferation, in prostate carcinoma. Am J Pathol 1996;149:765–73.[Abstract]
  14. Fornaro M, Manzotti M, Tallini G, et al. ß1C integrin in epithelial cells correlates with a nonproliferative phenotype: forced expression of ß1C inhibits prostate epithelial cell proliferation. Am J Pathol 1998;153:1079–87.[Abstract/Free Full Text]
  15. Fornaro M, Tallini G, Zheng DQ, Flanagan WM, Manzotti M, Languino LR. p27kip1 acts as a downstream effector of and is coexpressed with the ß1C integrin in prostatic adenocarcinoma. J Clin Invest 1999;103:321–9.[Medline]
  16. Manzotti M, Dell'Orto P, Maisonneuve P, Fornaro M, Languino LR, Viale G. Down-regulation of ß1C integrin in breast carcinomas correlates with high proliferative fraction, high histological grade, and larger size. Am J Pathol 2000;156:169–74.[Abstract/Free Full Text]
  17. Eliceiri BP. Integrin and growth factor receptor crosstalk. Circ Res 2001;89:1104–10.[Abstract/Free Full Text]
  18. Baserga R. The IGF-1 receptor in cancer research. Exp Cell Res 2003;253:1–6.
  19. Neudauer CL, McCarthy JB. Insulin-like growth factor I-stimulated melanoma cell migration requires phosphoinositide 3-kinase but not extracellular-regulated kinase activation. Exp Cell Res 2003;286:128–37.[CrossRef][Medline]
  20. Cardillo MR, Monti S, Di Silverio F, Gentile V, Sciarra F, Toscano V. Insulin-like growth factor (IGF)-I, IGF-II and IGF type I receptor (IGFR-I) expression in prostatic cancer. Anticancer Res 1996;23:3825–35.
  21. Tennant MK, Thrasher JB, Twomey PA, Drivdahl RH, Birnbaum RS, Plymate SR. Protein and messenger ribonucleic acid (mRNA) for the type 1 insulin-like growth factor (IGF) receptor is decreased and IGF-II mRNA is increased in human prostate carcinoma compared to benign prostate epithelium. J Clin Endocrinol Metab 1996;81:3774–82.[Abstract]
  22. Chott A, Sun Z, Morganstern D, et al. Tyrosine kinases expressed in vivo by human prostate cancer bone marrow metastases and loss of the type 1 insulin-like growth factor receptor. Am J Pathol 1999;155:1271–9.[Abstract/Free Full Text]
  23. Kaplan PJ, Mohan S, Cohen P, Foster BA, Greenberg NM. The insulin-like growth factor axis and prostate cancer: lessons from the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Cancer Res 1999;59:2203–9.[Abstract/Free Full Text]
  24. Nickerson T, Chang F, Lorimer D, Smeekens SP, Sawyers CL, Pollak M. In vivo progression of LAPC-9 and LNCaP prostate cancer models to androgen independence is associated with increased expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR). Cancer Res 2001;61:6276–80.[Abstract/Free Full Text]
  25. Hellawell GO, Turner GD, Davies DR, Poulsom R, Brewster SF, Macaulay VM. Expression of the type 1 insulin-like growth factor receptor is up-regulated in primary prostate cancer and commonly persists in metastatic disease. Cancer Res 2002;62:2942–50.[Abstract/Free Full Text]
  26. Holgado-Madruga M, Emlet DR, Moscatello DK, Godwin AK, Wong AJ. A Grb2-associated docking protein in EGF- and insulin-receptor signalling. Nature 1996;379:560–4.[CrossRef][Medline]
  27. Winnay JN, Bruning JC, Burks DJ, Kahn CR. Gab-1-mediated IGF-1 signaling in IRS-1-deficient 3T3 fibroblasts. J Biol Chem 2000;275:10545–50.[Abstract/Free Full Text]
  28. Gu H, Neel BG. The "Gab" in signal transduction. Trends Cell Biol 2003;13:122–30.[CrossRef][Medline]
  29. Goel HL, Fornaro M, Moro L, et al. Selective modulation of type 1 insulin-like growth factor receptor signaling and functions by ß1 integrins. J Cell Biol 1995;166:407–18.
  30. Kameda H, Risinger JI, Han BB, et al. Expression of Gab1 lacking the pleckstrin homology domain is associated with neoplastic progression. Mol Cell Biol 2001;21:6895–905.[Abstract/Free Full Text]
  31. Fornaro M, Lovecchio M, Jose P, Zheng D-Q, Moro L, Languino LR. Epitope-specific antibodies to the ß1C integrin cytoplasmic domain variant. Exp Mol Pathol 2001;70:275–80.[CrossRef][Medline]
  32. Mentor-Marcel R, Lamartiniere CA, Eltoum IE, Greenberg NM, Elgavish A. Genistein in the diet reduces the incidence of poorly differentiated prostatic adenocarcinoma in transgenic mice (TRAMP). Cancer Res 2001;61:6777–82.[Abstract/Free Full Text]
  33. Greenberg NM, DeMayo F, Finegold MJ, et al. Prostate cancer in a transgenic mouse. Proc Natl Acad Sci U S A 1995;92:3439–43.[Abstract/Free Full Text]
  34. Gingrich JR, Barrios RJ, Kattan MW, Nahm HS, Finegold MJ, Greenberg NM. Androgen-independent prostate cancer progression in the TRAMP model. Cancer Res 1997;57:4687–91.[Abstract/Free Full Text]
  35. Folkvord JM, Viders D, Coleman-Smith A, Clark RA. Optimization of immunohistochemical techniques to detect extracellular matrix proteins in fixed skin specimens. J Histochem Cytochem 1989;37:105–13.[Abstract]
  36. Shappell SB, Thomas GV, Roberts RL, et al. Prostate pathology of genetically engineered mice: definitions and classification. The consensus report from the Bar Harbor meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee. Cancer Res 2004;64:2270–305.[Abstract/Free Full Text]
  37. Wennerberg K, Lohikangas L, Gullberg D, Pfaff M, Johansson S, Fassler R. ß1 integrin-dependent and -independent polymerization of fibronectin. J Cell Biol 1996;132:227–38.[Abstract/Free Full Text]
  38. Manes T, Zheng DQ, Tognin S, Woodard AS, Marchisio PC, Languino LR. {alpha}vß3 integrin expression up-regulates cdc2, which modulates cell migration. J Cell Biol 2003;161:817–26.[Abstract/Free Full Text]
  39. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494–8.[CrossRef][Medline]
  40. Rubini M, Hongo A, D'Ambrosio C, Baserga R. The IGF-I receptor in mitogenesis and transformation of mouse embryo cells: role of receptor number. Exp Cell Res 1997;230:284–92.[CrossRef][Medline]
  41. Valentinis B, Baserga R. IGF-I receptor signalling in transformation and differentiation. Mol Pathol 2001;54:133–7.[Abstract/Free Full Text]
  42. Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 1993;75:59–72.[Medline]
  43. Sell C, Dumenil G, Deveaud C, et al. Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts. Mol Cell Biol 1994;14:3604–12.[Abstract/Free Full Text]
  44. Sell C, Rubini M, Rubin R, Liu JP, Efstratiadis A, Baserga R. Simian virus 40 large tumor antigen is unable to transform mouse embryonic fibroblasts lacking type 1 insulin-like growth factor receptor. Proc Natl Acad Sci U S A 1993;90:11217–21.[Abstract/Free Full Text]
  45. Parsons JT, Martin KH, Slack JK, Taylor JM, Weed SA. Focal adhesion kinase: a regulator of focal adhesion dynamics and cell movement. Oncogene 2000;19:5606–13.[CrossRef][Medline]
  46. Bloch W, Forsberg E, Lentini S, et al. ß1 integrin is essential for teratoma growth and angiogenesis. J Cell Biol 1997;139:265–78.[Abstract/Free Full Text]
  47. White DE, Kurpios NA, Zuo D, et al. Targeted disruption of ß1-integrin in a transgenic mouse model of human breast cancer reveals an essential role in mammary tumor induction. Cancer Cell 2004;6:159–70.[CrossRef][Medline]
  48. Reiss K, Wang JY, Romano G, Tu X, Peruzzi F, Baserga R. Mechanisms of regulation of cell adhesion and motility by insulin receptor substrate-1 in prostate cancer cells. Oncogene 2001;20:490–500.[CrossRef][Medline]
  49. Vuori K, Ruoslahti E. Association of insulin receptor substrate-1 with integrins. Science (Wash DC) 1994;266:1576–9.[Abstract/Free Full Text]
  50. Ling Y, Maile LA, Badley Clarke J, Clemmons DR. DOK1 mediates SHP-2 binding to the {alpha}Vß3 integrin and thereby regulates insulin-like growth factor I signaling in cultured vascular smooth muscle cells. J Biol Chem 2005;280:3151–8.[Abstract/Free Full Text]



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