Cancer Research Aziza Shad Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chung, J.
Right arrow Articles by Mercurio, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chung, J.
Right arrow Articles by Mercurio, A. M.
[Cancer Research 64, 4711-4716, July 15, 2004]
© 2004 American Association for Cancer Research

Advances in Brief

Hypoxia-Induced Vascular Endothelial Growth Factor Transcription and Protection from Apoptosis Are Dependent on {alpha}6ß1 Integrin in Breast Carcinoma Cells

Jun Chung, Sangoh Yoon, Kaustubh Datta, Robin E. Bachelder and Arthur M. Mercurio

Division of Cancer Biology and Angiogenesis, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
The {alpha}6ß1 integrin has been implicated in breast carcinoma progression, but the mechanisms involved remain elusive. MDA-MB-435 cells engineered to be deficient in {alpha}6ß1 expression form primary tumors that are highly apoptotic and unable to metastasize, although they exhibit no increased apoptosis in vitro under standard culture conditions. Based on the hypothesis that {alpha}6ß1 is necessary for the survival of these cells in the tumor microenvironment, we report here that hypoxia protects these cells from apoptosis induced by serum deprivation and that hypoxia-mediated protection requires {alpha}6ß1 expression. We investigated the influence of {alpha}6ß1 on vascular endothelial growth factor (VEGF) expression because autocrine VEGF is necessary for the survival of serum-deprived cells in hypoxia. The results obtained indicate that {alpha}6ß1 is necessary for VEGF expression because the ability of hypoxia to activate HIF-1 and to stimulate VEGF transcription in MDA-MB-435 cells is dependent on {alpha}6ß1 expression by a mechanism that involves protein kinase C-{alpha}.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
An important role for the {alpha}6ß1 integrin in breast cancer progression has been indicated by several studies (1, 2, 3) . The involvement of this integrin in progression was suggested first by the finding that high expression of the {alpha}6 subunit in women with breast cancer correlated significantly with reduced survival times (1) . In an analysis of 119 patients with invasive breast carcinoma, all of the patients with low or absent {alpha}6 expression survived, whereas the mortality rate of the patients with a high level of {alpha}6 expression was 19%. Of note, 30 of 34 of the patients that presented with distant metastases were highly positive for {alpha}6 expression. This study is consistent with the report that the metastatic potential of MDA-MB-435 cells correlates with their level of {alpha}6ß1 expression (3) . Moreover, when {alpha}6ß1-deficient MDA-MB-435 cells were inoculated into the mammary fat pads of nude mice, primary tumor size was significantly diminished compared with the parental cells because of increased apoptosis (2) . The {alpha}6ß1-deficient cells did not form metastases in the lung, as did the parental cells, because of their inability to survive in this organ (2) . Interestingly, these {alpha}6ß1-deficient cells did not differ in their ability to survive in vitro under standard culture conditions, suggesting that {alpha}6ß1 is needed for survival within the tumor microenvironment. Given that the microenvironment of solid tumors is often hypoxic and lacks the rich growth factor milieu present in culture medium, we examined the hypothesis that this integrin contributes to the survival of MDA-MB-435 cells in such conditions. Interestingly, the data obtained indicate that hypoxia protects these cells from apoptosis induced by serum deprivation and that this protection depends on {alpha}6ß1 expression. Protection from apoptosis under these conditions requires autocrine vascular endothelial growth factor (VEGF), and additional analysis revealed that {alpha}6ß1 is necessary for VEGF expression because it functions in concert with hypoxia to activate hypoxia inducible factor (HIF)-1 and to stimulate VEGF transcription by a mechanism that involves protein kinase C (PKC)-{alpha}.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
Cells and Reagents.
MDA-MB-435 breast carcinoma cells were obtained from the Lombardi Breast Cancer Depository at Georgetown University (Washington, DC). MDA-MB-435 cells that had been sorted by fluorescence-activated cell sorting to obtain populations that express relatively high or low {alpha}6ß1 expression were provided by Beth Israel Deaconess Medical Center (Boston, MA). Myrsitylated PKC-{alpha} and PKC-{epsilon} constructs were obtained from Dr. Alex Toker (Beth Israel Deaconess Medical Center). The HIF-1 dominant-negative mutant constructs A26E and K29E (4 , 5) were provided by Dr. Dev Mukhopadhyay (Beth Israel Deaconess Medical Center).

The following antibodies were used: an HIF-1{alpha} monoclonal antibody (Novus Biological, Newington, NH); a rabbit p300 polyclonal antibody (Upstate Biotechnology, Lake Placid, NY); an {alpha}6 integrin monoclonal antibody, clone 2B7 (prepared in our laboratory); and a rabbit actin antibody (Sigma, St. Louis, MO). Rabbit polyclonal anti-VEGF serum (clone 618) was obtained from Dr. Don Senger (Beth Israel Deaconess Medical Center).

Small Interfering RNA (siRNA) Experiments.
A siRNA specific for the {alpha}6 integrin subunit (GGUCGUGACAUGUGCUCAC) and a scrambled-sequence control (AUGCAGAGUGGCGCUCUCU) were synthesized by Dharmacon, Inc. (Lafayette, CO). A siRNA specific for {alpha}5 integrin subunit (UGGCUCAGACAUUCGAUCC) and control siRNA were synthesized by Qiagen, Inc. (Valencia, CA). Cells (1 x 105) were plated onto 35-mm tissue culture dishes a day before the transfection of 200 nM siRNA duplex with 25 mg of TransIT-TKO transfection reagent (Mirus, Madison, WI) in the presence of serum. A day after transfection, the transfection medium was aspirated from cells, and fresh complete medium was added and incubated for an additional 48–72 h. For each transfection, flow cytometry was used to assess {alpha}6 integrin expression.

VEGF Antisense Strategy.
Either a VEGF antisense 2'-O-methyl phosphorothioate oligodeoxynucleotide (5'-CACCCAAGACAGCAGAAG-3') or a VEGF sense 2'-O-methyl phosphorothioate oligodeoxynucleotide (5'-CTTTCTGCTGTCTTGGGTG) at a concentration of 0.3 µM was transfected into MDA-MB-435 cells as described previously (6) .

Protein Analysis.
For the analysis of protein expression, cells were extracted in radioimmunoprecipitation assay buffer [20 mM Tris buffer (pH 7.4) containing 0.14 M NaCl; 1% NP40; 10% glycerol; 1 mM sodium orthovanadate; 2 mM phenylmethylsulfonyl fluoride; and 5 µg/ml aprotinin, pepstatin, and leupeptin], and immunoblotting was performed as described previously (7) . For experiments that assessed HIF-1 expression and HIF-1 association with p300, nuclei were isolated and extracted as described previously (8 , 9) and used for immunoblotting and immunoprecipitation experiments.

Apoptosis Assay.
Adherent and nonadherent cells were harvested and assayed for apoptosis using annexin V-FITC (Biosource, Sunnyvale, CA) and propidium iodide (Biosource) as described previously (7) . In some experiments, cells were incubated in low serum [0.5% fetal bovine serum (FBS)] in hypoxia with or without recombinant VEGF165 (final concentration, 100 ng/ml; R&D Systems) along with 1 µg/ml heparin (Sigma) before the apoptosis assay.

Quantitative Real-Time PCR.
Quantitative analysis of VEGF mRNA expression was performed by real-time PCR as described previously (7) .

Transcription Assays.
Human VEGF promoters [either the full-length 2.6-kb promoter or 0.35-kb deletion mutant lacking the hypoxia response element (HRE)] were cloned into pGL3basic vector (Promega, Madison, WI) and used to assess VEGF promoter activity using firefly luciferase as the reporter gene (10) . Cells at a confluency of 85–95% were used for all experiments and incubated with 20 µM ZVAD-FMK (Promega) to prevent apoptosis during the experiment. Plasmids were transiently transfected with the Effectene transfection kit (Qiagen, Inc.) according to the manufacturer’s protocol. Thirty h after transfection, cells were washed with PBS and lysed with reporter buffer (Promega) at room temperature for 15 min followed by the luciferase assay. Luciferase activity was measured with a luminometer (MicroLumat LB96P; Berthold Technologies, Bad Wildbad, Germany) using the Dual-Glo luciferase assay kit (Promega). Renilla luciferase construct was used as an internal control to normalize the result. Triplicate readings were taken for each experiment, and SDs were calculated.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
Hypoxia Protects Serum-Deprived MDA-MB-435 Cells from Apoptosis: Role of {alpha}6ß1 Integrin.
To assess the importance of the {alpha}6ß1 integrin for the survival of tumor cells in stress conditions, we used MDA-MB-435 cells, which express {alpha}6ß1 but not {alpha}6ß4 (2 , 11) . Two approaches were used to modulate {alpha}6ß1 expression in these cells. In the first approach, cells were sorted by fluorescence-activated cell sorting to obtain two distinct clonal populations that exhibited either relatively high or low {alpha}6 integrin surface expression. As shown in Fig. 1ACitation , these populations differed by almost 4-fold in their mean fluorescence of {alpha}6 integrin expression. The second approach involved expression of a siRNA oligonucleotide specific for the {alpha}6 integrin subunit. Expression of this siRNA in MDA-MB-435 cells that expressed high levels of {alpha}6ß1 resulted in an approximate 70% reduction in the surface expression of this integrin (Fig. 1B)Citation . This siRNA did not alter expression of the {alpha}5 integrin subunit (Fig. 1B)Citation .



View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Expression of {alpha}6ß1 integrin is needed for protection from apoptosis by hypoxia. A, MDA-MB-435 cells that exhibited either high or low {alpha}6 expression were analyzed by flow cytometry, and data are reported as mean channel fluorescence (±SD) of {alpha}6 expression relative to a nonspecific IgG. B, siRNAs specific for {alpha}6 and a scrambled (scr) siRNA were transfected into cells that expressed high levels of {alpha}6, and cells were analyzed by flow cytometry 72 h later for {alpha}6 and {alpha}5 surface expression. Data are reported as in A. C, the cells characterized in A and B were incubated with either 10% FBS (+serum) or 0.5% FBS (–serum) in either normoxia (–) or hypoxia (+) for 24 h and stained with annexin V-FITC and propidium iodide (PI). Ab, antibody.

 
The cell populations described above were maintained in either a normoxic or hypoxic environment in the presence of either 10 or 0.5% FBS for 24 h, and apoptosis was assessed by annexin-FITC staining (Fig. 1C)Citation . In normoxia, serum deprivation (0.5% FBS) increased the level of apoptosis by approximately 3–4-fold compared with cells maintained in 10% serum (Fig. 1C)Citation . Hypoxia, however, protected cells from apoptosis induced by serum deprivation as a function of {alpha}6ß1 expression. More specifically, both the low-expressing {alpha}6 cells and the {alpha}6 siRNA-treated cells were significantly more apoptotic under these conditions than were the corresponding high-{alpha}6ß1-expressing control cells (Fig. 1C)Citation . To assess the integrin specificity of these results, we used a siRNA to reduce expression of the {alpha}5ß1 integrin, which is expressed in MDA-MB-435 cells at levels comparable with {alpha}6ß1. As shown in Fig. 2ACitation , expression of a siRNA specific for the {alpha}5 integrin subunit reduced surface expression of {alpha}5ß1 by approximately 30%. This reduction in {alpha}5ß1 expression, however, had no impact on apoptosis induced by serum deprivation under either normoxic or hypoxic conditions as assessed by annexin-V-FITC staining (Fig. 2B)Citation .



View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Expression of {alpha}6ß1 but not {alpha}5ß1 integrin is needed for protection from apoptosis by hypoxia. A, cells that express high levels of {alpha}6ß1 were transfected with siRNAs specific for {alpha}6 or {alpha}5 or with scrambled sequences (scr). After 72 h, integrin expression was assessed by flow cytometry as in Fig. 1ACitation . B, the populations of cells described above were assessed for apoptosis under the conditions described in Fig. 1CCitation . Apoptosis is reported as the percentage of annexin V-FITC+, propidium iodide (PI–) cells. The data shown are mean values (±SD) of a representative experiment performed in triplicate. Ab, antibody.

 
Hypoxic Stimulation of VEGF Transcription Is Dependent on {alpha}6ß1.
Given the recent reports that autocrine VEGF is necessary for the survival of breast and other carcinoma cells (6 , 7 , 12, 13, 14, 15) , we assessed whether the ability of hypoxia to prevent apoptosis is VEGF dependent. As shown in Fig. 3ACitation , expression of an antisense VEGF oligonucleotide in high-{alpha}6-expressing cells reduced hypoxia-induced VEGF expression by approximately 60% in comparison with the sense oligonucleotide, and it increased the apoptosis of serum-deprived cells in hypoxia 2-fold. Furthermore, incubation of low-{alpha}6-expressing cells with exogenous recombinant VEGF165 inhibited the apoptosis induced by serum deprivation significantly (Fig. 3A)Citation . These findings led us to examine the hypothesis that {alpha}6ß1 influences hypoxia-induced VEGF expression. In the presence of 10% serum, VEGF expression is relatively high and independent of {alpha}6ß1 expression in MDA-MB-435 cells (data not shown). Serum deprivation for 24 h, however, reduced VEGF expression to nearly undetectable levels in normoxia (Fig. 3B)Citation . In contrast, hypoxia sustained both total VEGF and secreted VEGF as a function of {alpha}6ß1 expression (Fig. 3B)Citation . These data indicate that serum induces VEGF expression independently of {alpha}6ß1, but in the absence of serum, VEGF expression decreases and apoptosis increases. Hypoxia prevents this increase in apoptosis in serum-deprived cells by inducing VEGF expression in an {alpha}6ß1-dependent manner.



View larger version (36K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. {alpha}6ß1 integrin is necessary for hypoxia-induced VEGF expression. A, MDA-MB-435 cells expressing high {alpha}6 integrin were transfected with either the VEGF sense (S) or antisense (AS) oligonucleotide and recovered in complete medium overnight, followed by incubation in low serum (0.5% FBS) either in normoxia (N) or hypoxia (H) for 24 h. In addition, cells expressing low {alpha}6 integrin were incubated in low serum (0.5% FBS) in hypoxia with or without recombinant VEGF165 (rVEGF; final concentration, 100 ng/ml; R&D Systems) along with 1 µg/ml heparin. The level of apoptosis is indicated as the percentage of annexin V-FITC+, propidium iodide (PI–) cells. Extracts of these cells were analyzed for their relative expression of VEGF and actin by immunoblotting. B, extracts of the cells described in Fig. 1CCitation were analyzed for their relative expression of VEGF and actin by immunoblotting (total VEGF). Culture media from the cells described in Fig. 1CCitation were concentrated and analyzed for VEGF expression by immunoblotting (secreted VEGF). C, VEGF mRNA was quantified by real-time PCR in the cells described in Fig. 1CCitation that had been maintained in either normoxia or hypoxia for 24 h in the presence of low serum. Data are presented as the mean ratio of VEGF to ß-actin mRNA (±SD). D, a luciferase construct conjugated to the VEGF promoter was transfected into the cells described in Fig. 1CCitation . Luciferase activity (±SD) was assayed after the cells had been maintained in either normoxia or hypoxia for 24 h in the presence of low serum. scr, scrambled.

 
To address the mechanism by which {alpha}6ß1 influences hypoxia-induced VEGF expression, we quantified VEGF mRNA expression by real-time PCR. Hypoxia increased VEGF mRNA expression approximately 2-fold in serum-deprived cells with high {alpha}6ß1 expression but not in cells with reduced expression of this integrin (Fig. 3C)Citation . This finding prompted us to evaluate the mechanism by which this integrin contributes to VEGF transcription. For this purpose, cells were transfected with a reporter construct that consisted of the intact VEGF promoter conjugated to luciferase. The relative luminescence of the reporter construct increased approximately 3-fold in hypoxia but only in those populations of cells that expressed high levels of {alpha}6ß1 (Fig. 3D)Citation . The ability of hypoxia to increase VEGF promoter activity in cells selected for low {alpha}6ß1 expression or treated with the {alpha}6 siRNA was negligible (Fig. 3D)Citation . To exclude the possibility that the apoptosis induced by these conditions affects VEGF promoter activity, cells were incubated with the general caspase inhibitor ZVAD-FMK. The use of this inhibitor prevented the apoptosis triggered by serum deprivation and low {alpha}6 expression (data not shown). The effect of {alpha}6ß1 on VEGF transcription appears to be independent of its ligation because we observed comparable VEGF expression when cells were plated on either laminin, an {alpha}6ß1 ligand, or other matrix proteins that are not ligands for this integrin such as collagen (data not shown).

MDA-MB-435 cells lack expression of VEGFR-1 (flt-1) and VEGFR-2 (KDR, flk-1; Refs. 16 and 17 ). However, they express neuropilin-1 (17) , and the expression of this VEGF receptor did not differ between high- and low-a6-expressing cells, as evidenced by immunoblotting (data not shown) suggesting that the {alpha}6ß1 integrin does not influence the expression of VEGF receptors.

The {alpha}6ß1 Integrin Regulates the Transcriptional Activity of HIF-1.
We examined the hypoxic induction of HIF-1{alpha} expression as a function of {alpha}6ß1 expression because HIF-1 plays a pivotal role in stimulating the transcription of VEGF and many other genes in hypoxia (18) . Interestingly, as shown in Fig. 4ACitation , the level of HIF-1{alpha} induction in hypoxia was comparable between the high and low {alpha}6 clones, suggesting that {alpha}6ß1 integrin is not involved in either the expression or stabilization of HIF-1{alpha}. To assess HIF-1 activation directly, we monitored the association of HIF-1 with its coactivator, p300 (Fig. 4A)Citation . HIF-1 binding to HRE itself is not sufficient to activate target gene transcription, and HIF-1 association with p300 is required to recruit the RNA polymerase II complex to initiate transcription (19) . Therefore, association of HIF-1 with p300 is an indicator of HIF-1 activation. Association of HIF-1 with p300 was maximal at 6 h in cells that expressed high levels of {alpha}6ß1 and diminished by 24 h (Fig. 4A)Citation . In cells that expressed low levels of {alpha}6ß1, however, association of HIF-1 with p300 was barely detectable (Fig. 4A)Citation . The total level of p300 expression is similar in both populations of cells (Fig. 4A)Citation . These data indicate that the {alpha}6ß1 integrin influences HIF-1 activation but not its expression.



View larger version (46K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. {alpha}6ß1 is involved in HIF-1 activation via PKC-{alpha}. A, HIF-1{alpha} expression was assessed by immunoblotting nuclear extracts obtained from cells that expressed either high or low levels of {alpha}6 and that had been maintained in either normoxia for 24 h or hypoxia for 6 or 24 h in the presence of low serum. p300 was immunoprecipitated from the nuclear extracts described above. The immunoprecipitates were analyzed by immunoblotting for p300 and HIF-1{alpha} expression. B, luciferase constructs conjugated to either the full-length promoter (2.6 kb) or a deletion mutant lacking the HRE (0.35 kb) were transfected into cells that expressed high levels of {alpha}6 integrin. Dominant-negative HIF-1 constructs (A26E and K29E) were cotransfected with a luciferase construct conjugated with the full-length promoter (2.6 kb) into MDA-MB-435 cells that expressed high levels of {alpha}6 integrin. VEGF promoter activity was assessed by the luciferase assay as described in Fig. 3DCitation . C, cells that express high levels of {alpha}6ß1 were incubated with 0.5% FBS for 6 h in normoxia or hypoxia with or without 1 µM Gö6976. HIF-1{alpha} expression and association with p300 were assessed as described above. D, cells that express low levels of {alpha}6ß1 were transfected with a constitutively active PKC-{alpha} construct (Lane 3), a constitutively active PKC-{epsilon} construct (Lane 4), or an empty vector (Lanes 1 and 2) and analyzed for HIF-1{alpha} and p300 association in hypoxia as described above. Cells that express high levels of {alpha}6ß1 were used as positive control (Lanes 5 and 6).

 
To confirm the involvement of HIF-1 in VEGF transcription under hypoxia, we used a luciferase construct conjugated with a VEGF promoter (0.35 kb) that lacks the HRE (Fig. 4B)Citation . We also expressed HIF-1 dominant-negative mutants that block HIF-1 binding to HRE. Expression of this mutant promoter, as well as the two different dominant-negative HIF-1 mutants, in high-{alpha}6-expressing cells level did not result in hypoxia-induced increase in VEGF promoter activity (Fig. 4B)Citation , indicating that HIF-1 is playing a major role in {alpha}6ß1-mediated VEGF transcription in hypoxia.

Our consideration of possible mechanisms involved in {alpha}6ß1 regulation of HIF-1 activation led us to PKC because of the report that this integrin associates with specific PKC isoforms (20) and other studies that have linked PKCs to HIF-1 activation, VEGF expression, apoptosis, and survival signaling (21, 22, 23, 24) . Pharmacological inhibition of PKC-{alpha} activity using Gö6976 prevented {alpha}6ß1-mediated HIF-1 activation as measured by p300 association (Fig. 4C)Citation . The finding that PKC-{alpha} activity is necessary for HIF-1 activation raised the possibility that HIF-1 activation could be induced in cells that expressed low levels of {alpha}6ß1 by expression of a constitutively active form of PKC-{alpha}. Indeed, as shown in Fig. 4CCitation , expression of a constitutively active form of PKC-{alpha} increased HIF-1 activity dramatically in cells that normally could not activate HIF-1 under hypoxia (Fig. 4A)Citation . Expression of a constitutively active form of PKC-{epsilon}, in contrast, did not rescue the ability of these cells to activate HIF-1 under hypoxia (Fig. 4D)Citation . Collectively, these data highlight a key role for PKC-{alpha} in HIF-1 activation and VEGF transcription.

To date, the mechanism of HIF-1 activation has focused on two different posttranslational modifications at the COOH-terminal activation domain of HIF-1{alpha} that are known to regulate the association of p300 with HIF-1{alpha}. One modification is the hydroxylation of a specific asparagine residue (amino acid 803 of HIF-1{alpha}) at the COOH-terminal activation domain that prevents the association of p300 with HIF-1 in normoxia (25 , 26) . The other modification is the phosphorylation of a threonine residue (amino acid 844 of HIF-1{alpha}) that is required for their association (27) . An important issue that arises, therefore, is how PKC-{alpha} facilitates HIF-1 activation and how this signaling pathway is linked to these other signaling pathways that have been implicated in HIF-1 activation. Our data indicate that PKC-{alpha} regulates the association of HIF-1 and p300 in hypoxia, which excludes a role for PKC-{alpha} in regulating asparagine hydroxylase because it is inactive in hypoxia. More likely, PKC-{alpha} is directly or indirectly involved in COOH-terminal activation domain phosphorylation to activate HIF-1.

Overall, our studies highlight a potential function for the {alpha}6ß1 integrin in stimulating VEGF transcription and providing a selective survival advantage for carcinoma cells in the tumor microenvironment in which both nutrients and oxygen supply are limited. Moreover, these findings reveal a mechanism that could account for the involvement of the {alpha}6ß1 integrin in tumor survival that has been observed in vivo, and they are consistent with the finding reported here and elsewhere (2 , 3) that breast carcinoma cells depend upon autocrine VEGF for survival. These findings may also bear on other aspects of VEGF function in cancer, as well as other targets of VEGF transcription that could contribute to tumor survival. The best-characterized functions of VEGF, produced by both tumor and stromal cells, are to increase vascular permeability (28) and to stimulate angiogenesis (18 , 29) . Thus, it can be inferred that the {alpha}6 integrin influence on VEGF transcription will also impact these core VEGF functions. In a different direction, HIF-1 is known to regulate the transcription of more than 40 target genes, many of which can contribute to the survival of tumor cells in hypoxia (30) . For this reason, it is conceivable that such genes function in concert with VEGF and {alpha}6ß1 to affect tumor survival. In addition, our data highlight a role for PKC-{alpha} in HIF-1 activation and VEGF transcription.


   ACKNOWLEDGMENTS
 
We thank Drs. Don Senger, Elizabeth Lipscomb, and Leslie Shaw for helpful insight and discussion. We also thank Dr. Dev Mukhopadhyay for providing reagents and technical assistance.


   FOOTNOTES
 
Grant support: NIH Grant CA89209 (A. Mercurio) and DAMD Grants BC001077 (J. Chung) and BC000697 (A. Mercurio).

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.

Requests for reprints: Arthur M. Mercurio, Beth Israel Deaconess Medical Center, Research North, 330 Brookline Avenue, Boston, MA 02215. Phone: (617) 667-7714; Fax: (617) 667-5531; E-mail: amercuri{at}caregroup.harvard.edu

Received 2/ 3/04. Revised 4/28/04. Accepted 5/24/04.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

  1. Friedrichs K, Ruiz P, Franke F, Gille I, Terpe HJ, Imhof BA. High expression level of {alpha}6 integrin in human breast carcinoma is correlated with reduced survival. Cancer Res., 55: 901-6, 1995.[Abstract/Free Full Text]
  2. Wewer UM, Shaw LM, Albrechtsen R, Mercurio AM. The integrin {alpha} 6 ß 1 promotes the survival of metastatic human breast carcinoma cells in mice. Am J Pathol, 151: 1191-8, 1997.[Abstract]
  3. Mukhopadhyay R, Theriault RL, Price JE. Increased levels of {alpha}6 integrins are associated with the metastatic phenotype of human breast cancer cells. Clin Exp Metastasis, 17: 325-32, 1999.[Medline]
  4. Michel G, Minet E, Ernest I, et al A model for the complex between the hypoxia-inducible factor-1 (HIF-1) and its consensus DNA sequence. J Biomol Struct Dyn, 18: 169-79, 2000.[Medline]
  5. Michel G, Minet E, Mottet D, Remacle J, Michiels C. Site-directed mutagenesis studies of the hypoxia-inducible factor-1{alpha} DNA-binding domain. Biochim Biophys Acta, 1578: 73-83, 2002.[Medline]
  6. Bachelder RE, Crago A, Chung J, et al Vascular endothelial growth factor is an autocrine survival factor for neuropilin-expressing breast carcinoma cells. Cancer Res., 61: 5736-40, 2001.[Abstract/Free Full Text]
  7. Chung J, Bachelder RE, Lipscomb EA, Shaw LM, Mercurio AM. Integrin ({alpha} 6 ß 4) regulation of eIF-4E activity and VEGF translation: a survival mechanism for carcinoma cells. J Cell Biol, 158: 165-74, 2002.[Abstract/Free Full Text]
  8. Graven KK, Farber HW. Endothelial cell hypoxic stress proteins. J Lab Clin Med, 132: 456-63, 1998.[CrossRef][Medline]
  9. Clegg JS, Jackson SA, Liang P, MacRae TH. Nuclear-cytoplasmic translocations of protein p26 during aerobic-anoxic transitions in embryos of Artemia franciscana. Exp Cell Res., 219: 1-7, 1995.[CrossRef][Medline]
  10. Pal S, Datta K, Khosravi-Far R, Mukhopadhyay D. Role of protein kinase Czeta in Ras-mediated transcriptional activation of vascular permeability factor/vascular endothelial growth factor expression. J Biol Chem, 276: 2395-403, 2001.[Abstract/Free Full Text]
  11. Shaw LM, Chao C, Wewer UM, Mercurio AM. Function of the integrin {alpha} 6 ß 1 in metastatic breast carcinoma cells assessed by expression of a dominant-negative receptor. Cancer Res., 56: 959-63, 1996.[Abstract/Free Full Text]
  12. Mercurio AM, Bachelder RE, Bates RC, Chung J. Autocrine signaling in carcinoma: VEGF and the {alpha}6ß4 integrin. Semin Cancer Biol, 14: 115-22, 2004.[CrossRef][Medline]
  13. Bates RC, Goldsmith JD, Bachelder RE, et al Flt-1-dependent survival characterizes the epithelial-mesenchymal transition of colonic organoids. Curr Biol, 13: 1721-7, 2003.[CrossRef][Medline]
  14. Foster RR, Hole R, Anderson K, et al Functional evidence that vascular endothelial growth factor may act as an autocrine factor on human podocytes. Am J Physiol Renal Physiol, 284: F1263-73, 2003.[Abstract/Free Full Text]
  15. Baek JH, Jang JE, Kang CM, Chung HY, Kim ND, Kim KW. Hypoxia-induced VEGF enhances tumor survivability via suppression of serum deprivation-induced apoptosis. Oncogene, 19: 4621-31, 2000.[CrossRef][Medline]
  16. Soker S, Fidder H, Neufeld G, Klagsbrun M. Characterization of novel vascular endothelial growth factor (VEGF) receptors on tumor cells that bind VEGF165 via its exon 7-encoded domain. J Biol Chem, 271: 5761-7, 1996.[Abstract/Free Full Text]
  17. Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell, 92: 735-45, 1998.[CrossRef][Medline]
  18. Semenza GL. HIF-1: using two hands to flip the angiogenic switch. Cancer Metastasis Rev, 19: 59-65, 2000.[CrossRef][Medline]
  19. Minet E, Michel G, Mottet D, Raes M, Michiels C. Transduction pathways involved in Hypoxia-Inducible Factor-1 phosphorylation and activation. Free Radic Biol Med, 31: 847-55, 2001.[CrossRef][Medline]
  20. Zhang XA, Bontrager AL, Hemler ME. Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific ß(1) integrins. J Biol Chem, 276: 25005-13, 2001.[Abstract/Free Full Text]
  21. Datta K, Li J, Bhattacharya R, Gasparian L, Wang E, Mukhopadhyay D. Protein kinase C {zeta} transactivates hypoxia-inducible factor {alpha} by promoting its association with p300 in renal cancer. Cancer Res., 64: 456-62, 2004.[Abstract/Free Full Text]
  22. Wu G, Mannam AP, Wu J, et al Hypoxia induces myocyte-dependent COX-2 regulation in endothelial cells: role of VEGF. Am J Physiol Heart Circ Physiol, 285: H2420-9, 2003.[Abstract/Free Full Text]
  23. O’Brian CA. Protein kinase C-{alpha}: a novel target for the therapy of androgen-independent prostate cancer?. Oncol Rep, 5: 305-9, 1998.[Medline]
  24. Gutcher I, Webb PR, Anderson NG. The isoform-specific regulation of apoptosis by protein kinase C. Cell Mol Life Sci, 60: 1061-70, 2003.[Medline]
  25. McNeill LA, Hewitson KS, Claridge TD, Seibel JF, Horsfall LE, Schofield CJ. Hypoxia-inducible factor asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the ß-carbon of asparagine-803. Biochem J, 367: 571-5, 2002.[CrossRef][Medline]
  26. Freedman SJ, Sun ZY, Poy F, et al Structural basis for recruitment of CBP/p300 by hypoxia-inducible factor-1 {alpha}. Proc Natl Acad Sci USA, 99: 5367-72, 2002.[Abstract/Free Full Text]
  27. Gradin K, Takasaki C, Fujii-Kuriyama Y, Sogawa K. The transcriptional activation function of the HIF-like factor requires phosphorylation at a conserved threonine. J Biol Chem, 277: 23508-14, 2002.[Abstract/Free Full Text]
  28. Dvorak HF, Nagy JA, Feng D, Brown LF, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis. Curr Top Microbiol Immunol, 237: 97-132, 1999.[Medline]
  29. Richard DE, Berra E, Pouyssegur J. Angiogenesis: how a tumor adapts to hypoxia. Biochem Biophys Res Commun, 266: 718-22, 1999.[CrossRef][Medline]
  30. Semenza G. Signal transduction to hypoxia-inducible factor 1. Biochem Pharmacol, 64: 993-8, 2002.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
E. Delamarre, S. Taboubi, S. Mathieu, C. Berenguer, V. Rigot, J.-C. Lissitzky, D. Figarella-Branger, L. Ouafik, and J. Luis
Expression of Integrin {alpha}6{beta}1 Enhances Tumorigenesis in Glioma Cells
Am. J. Pathol., August 1, 2009; 175(2): 844 - 855.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
C. Fischbach, H. J. Kong, S. X. Hsiong, M. B. Evangelista, W. Yuen, and D. J. Mooney
Cancer cell angiogenic capability is regulated by 3D culture and integrin engagement
PNAS, January 13, 2009; 106(2): 399 - 404.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. Spangenberg, E. U. Lausch, T. M. Trost, D. Prawitt, A. May, R. Keppler, S. A. Fees, D. Reutzel, C. Bell, S. Schmitt, et al.
ERBB2-Mediated Transcriptional Up-regulation of the {alpha}5{beta}1 Integrin Fibronectin Receptor Promotes Tumor Cell Survival Under Adverse Conditions.
Cancer Res., April 1, 2006; 66(7): 3715 - 3725.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. L. Goel, L. Moro, M. King, N. Teider, M. Centrella, T. L. McCarthy, M. Holgado-Madruga, A. J. Wong, E. Marra, and L. R. Languino
{beta}1 Integrins Modulate Cell Adhesion by Regulating Insulin-Like Growth Factor-II Levels in the Microenvironment
Cancer Res., January 1, 2006; 66(1): 331 - 342.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
I. Kryczek, A. Lange, P. Mottram, X. Alvarez, P. Cheng, M. Hogan, L. Moons, S. Wei, L. Zou, V. Machelon, et al.
CXCL12 and Vascular Endothelial Growth Factor Synergistically Induce Neoangiogenesis in Human Ovarian Cancers
Cancer Res., January 15, 2005; 65(2): 465 - 472.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chung, J.
Right arrow Articles by Mercurio, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chung, J.
Right arrow Articles by Mercurio, A. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online