Cancer Research Annual Meeting 2010 Protein Translation and Cancer
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Cancer Research 68, 6127, August 1, 2008. doi: 10.1158/0008-5472.CAN-08-1395
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Loss of Integrin {alpha}1β1 Ameliorates Kras-Induced Lung Cancer

Ines Macias-Perez1,2, Corina Borza1, Xiwu Chen1, Xuexian Yan1, Raquel Ibanez1, Glenda Mernaugh1, Lynn M. Matrisian2, Roy Zent1,2,3,4 and Ambra Pozzi1,2,4

Departments of 1 Medicine (Division of Nephrology), 2 Cancer Biology, and 3 Cell Biology, Vanderbilt University Medical Center, and 4 Department of Medicine, Veterans Affairs Hospital, Nashville, Tennessee

Requests for reprints: Ambra Pozzi, Department of Medicine, Division of Nephrology, Vanderbilt University, Medical Center North, B3115, Nashville, TN 37232. Phone: 615-322-4637; Fax: 615-322-4690; E-mail: ambra.pozzi{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The collagen IV binding receptor integrin {alpha}1β1 has been shown to regulate lung cancer due to its proangiogenic properties; however, it is unclear whether this receptor also plays a direct role in promoting primary lung tumors. To investigate this possibility, integrin {alpha}1-null mice were crossed with KrasLA2 mice that carry an oncogenic mutation of the Kras gene (G12D) and develop spontaneous primary tumors with features of non–small cell lung cancer. We provide evidence that KrasLA2/{alpha}1-null mice have a decreased incidence of primary lung tumors and longer survival compared with KrasLA2/{alpha}1 wild-type controls. Tumors from KrasLA2/{alpha}1-null mice were also smaller, less vascularized, and exhibited reduced cell proliferation and increased apoptosis, as determined by proliferating cell nuclear antigen and terminal deoxynucleotidyl-transferase–mediated dUTP nick-end staining, respectively. Moreover, tumors from the KrasLA2/{alpha}1-null mice showed diminished extracellular signal-regulated kinase (ERK) but enhanced p38 mitogen-activated protein kinase activation. Primary lung tumor epithelial cells isolated from KrasLA2/{alpha}1-null mice showed a significant decrease in anchorage-independent colony formation, collagen-mediated cell proliferation, ERK activation, and, most importantly, tumorigenicity when injected into nude mice compared with KrasLA2/{alpha}1 wild-type tumor cells. These results indicate that loss of the integrin {alpha}1 subunit decreases the incidence and growth of lung epithelial tumors initiated by oncogenic Kras, suggesting that both Kras and integrin {alpha}1β1 cooperate to drive the growth of non–small cell lung cancer in vivo. [Cancer Res 2008;68(15):6127–35]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Integrins are extracellular matrix receptors composed of noncovalently associated {alpha} and β chains (1, 2). In addition to their function as anchoring molecules, integrins transmit bidirectional signals that regulate many important aspects of cell behavior including proliferation, migration, differentiation, and survival (2). Of the 24 heterodimers identified thus far, the integrin family contains 4 major collagen receptors, namely integrins {alpha}1β1, {alpha}2β1, {alpha}10β1, and {alpha}11β1 (3–6). Integrin {alpha}1β1 is the principal collagen IV receptor, and upon engagement by this ligand, it regulates many cell functions, such as support of cell proliferation and survival (7) as well as synthesis of collagen (8), matrix metalloproteinase (MMPs; refs. 8–10), and reactive oxygen species (11).

The contribution of integrin {alpha}1β1 in tumor formation and progression is poorly defined. Using xenograft and orthotopic models of cancer, we showed that integrin {alpha}1-null ({alpha}1KO) mice develop less, smaller, and poorly vascularized tumors compared with WT mice. The reduced angiogenesis in the {alpha}1-null mice is due to increased levels of MMP9, which generates angiostatin with consequent inhibition of endothelial cell growth (9, 10, 12, 13). Thus, integrin {alpha}1β1 serves as a proangiogenic receptor, and loss of its expression by endothelial cells protects the host from tumor growth by reducing pathologic angiogenesis.

It is unclear whether integrin {alpha}1β1 plays a direct role in cancer initiation and growth. Observational studies linking expression of this receptor to cancer are highly variable and inconclusive. In this context, increased expression of integrin {alpha}1β1 in melanoma can either correlate with enhanced progression/metastasis (14, 15) as well with a favorable outcome (16). Integrin {alpha}1β1 has also been implicated in pulmonary neoplasms and its expression is up-regulated in bronchoalveolar carcinomas and pulmonary carcinoids (17). In contrast, down-regulation of this receptor was observed in malignant breast tumors (18), whereas no differences in its expression were detected between normal liver tissues and hepatomas or hepatocarcimomas (19). Similar controversies with respect to the contribution of integrin {alpha}1β1 in regulating tumor cell adhesion, migration, and invasion have been documented in vitro. For example, integrin {alpha}1β1 contributes to tumor cell invasion by inducing MMP3 expression in mouse mammary carcinoma cells (20), whereas in human gastric carcinoma cells, its expression prevents growth factor–induced tumor cell migration (21).

Integrins can activate multiple intracellular signaling upon ligand binding, including the Ras pathways. Integrin {alpha}vβ3- and {alpha}6β4-mediated Ras activation is necessary to support cell proliferation (22, 23). Integrin {alpha}1β1 is linked to Ras signaling via the adaptor protein Src homology (Shc) and collagen (24) and supports cell survival via the Shc/growth factor receptor binding protein 2 (Grb2)/extracellular signal-regulated kinase (ERK) axis (7). Given that Ras family members are proto-oncogenes and integrin {alpha}1β1 can activate the RAS–mitogen-activated protein kinase (MAPK) pathway (7, 24), it is conceivable that {alpha}1β1 cooperates with the Ras signaling in regulating tumorigenesis. To test this hypothesis, {alpha}1KO mice were crossed to the KrasLA2 mouse, a model of spontaneous non–small cell lung cancer (NSCLC). The KrasLA2 mice harbor a latent oncogenic G12D mutation in the Kras gene that is expressed only after somatic recombination (25). Mice carrying the mutated Kras allele have a decreased life span and develop spontaneous lung tumors as early as 5 days after birth (25). We show that the KrasLA2/{alpha}1KO mice have a decreased incidence, number, and size of primary lung tumors resulting in longer survival than KrasLA2/{alpha}1WT controls. Moreover, primary lung tumor epithelial cells isolated from KrasLA2/{alpha}1KO mice show decreased anchorage-independent colony formation, collagen-mediated cell growth, and, most importantly, tumorigenicity when injected into nude mice compared with KrasLA2/{alpha}1WT tumor cells. Thus, integrin {alpha}1β1 plays a key role in both the development and growth of lung cancer that is independent of integrin {alpha}1β1-mediated angiogenesis. Furthermore, these data suggest that this collagen receptor and oncogenic Kras cooperate to drive lung tumorigenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
The KrasLA mouse model of lung cancer. KrasLA2 mice (gift of Dr. T. Jacks, Massachusetts Institute of Technology, Cambridge, MA; ref. 25) were crossed with wild-type (WT) or {alpha}1KO mice on the SV129 background (generated as described in ref. 6) to obtain KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice. Because the K-rasLA allele is nonfunctional in the germline configuration (25), the mice were maintained in a heterozygous state, as described (25). Age- and sex-matched KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO were sacrificed at different time points ranging from ages 7 to 250 d as these mice develop multiple lung tumors as early as 1 wk after birth and do not usually survive >200 d (25). Representative analysis of KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice sacrificed 120 d after birth are shown. The lungs were removed immediately at sacrifice, and the number of tumors visible on the lung surface were counted (36 KrasLA2/{alpha}1WT and 24 KrasLA2/{alpha}1KO mice were analyzed). Tumor diameter was measured with a caliper, and tumors were divided into three groups: 0 to 2 mm, 2 to 5 mm, and >5 mm tumor diameter (170 tumors from 14 KrasLA2/{alpha}1WT and 102 tumors from 24 KrasLA2/{alpha}1KO mice were used for analysis). Parts of lungs containing tumors were fixed in 4% paraformaldehyde and processed for histologic analysis or immunohistochemistry.

Survival analysis. KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice were analyzed daily from birth and sacrificed at the first sign of shortness of breath, reduced locomotion, and reduced body weight (>20% total body weight). Lungs were removed and analyzed for the presence of visible tumors on the lung surface. Only mice with visible lung tumors were used for the survival experiment. Twenty-five mice per genotype were used for analysis.

Immunostaining and quantification. Immunohistochemistry on lung paraffin sections (5 µm each) was performed using rat anti-mouse CD31 (1:100; PharMingen), rabbit anti-mouse proliferating cell nuclear antigen (PCNA; 1:100; Santa Cruz), rabbit anti-mouse phospho-ERK (1:200; Cell Signaling), or mouse anti-mouse phospho-p38 MAPK (1:200; Cell Signaling) followed by the appropriate HRP-conjugated secondary antibodies (1:200; Jackson) and Sigma Fast 3,3'-diaminobenzidine (DAB) chromogenic tablets (Sigma). CD31-positive structures within tumors were recorded (3 images per tumor with 4 tumors per lung with a total of 10 lungs per genotype) and processed as previously described (9). Tumor vascularity was expressed as percentage of area occupied by CD31-positive structures per microscopic field.

Tumor apoptosis was evaluated by staining paraffin sections with the Dead End colorimetric terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling (TUNEL) system (Promega) using DAB as chromogenic substrate. Quantification of proliferating and apoptotic cells was done by expressing the number of PCNA or apoptotic-positive cells (3 images per tumor with 4 tumors per lung with a total of 10 lungs per genotype) per microscopic field (x400).

Quantification of phospho-ERK and phospho-p38 MAPK–positive cells was performed as indicated above. Both and phospho-ERK or p38-positive cells within a microscopic field were evaluated and expressed; the result is expressed as a percentage of total ERK or p38-positive cells per microscopic field.

Isolation of lung tumor cells from KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice. Age-matched KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO male mice were sacrificed, and the lungs were perfused with PBS. Lungs were then removed, tumors were excised, minced, and incubated with 0.25% trypsin/1 mmol/L EDTA for 10 min at 37°C. After brief centrifugation, the digested supernatant was quickly removed, incubated with an equal volume of DMEM/10% FCS, and subsequently spun for 5 min at 1,000 rpm. The cell pellet was resuspended in DMEM/10% FCS and cultured in a 6-well plate. Fresh trypsin/EDTA was added to the leftover digested tumor tissues, and the process above was repeated five times. When the lung epithelial cells reached confluence, they were disaggregated with 0.25% Trypsin/EDTA and subcultured using standard technique. To remove tumor-associated fibroblasts, cell cultures were subjected to several rounds of trypsinization and the medium was changed to bronchial epithelial growth medium supplemented with rhEGF, insulin, hydrocortisone, transferrin, retinoic acid, epinephrine, and triiodothyronine (Clonetics) until no further fibroblasts were observed. After cultures of lung epithelial cells showed stable growth patterns, they were maintained in DMEM supplemented with 10% FCS. Three preparations of tumor cells were performed. Each preparation was derived from tumors pooled from 6 to 9 KrasLA2/{alpha}1WT and 8 to 10 KrasLA2/{alpha}1KO mice. As each of the three preparations led to similar results with respect to adhesion, signaling, anchorage-independent, and -dependent growth, we only show the results obtained from one representative batch.

Transfection of the integrin {alpha}1 subunit into KrasLA2/{alpha}1KO cells. The human integrin {alpha}1 cDNA (gift of Dr. E. Marcantonio, Columbia University, New York, NY) was subcloned into pAdeno-X (Clontech) following manufacturer instructions. Empty adenovirus (AdVo) and adenovirus expressing the integrin {alpha}1 subunit (Ad{alpha}1) were transfected in HEK cells, amplified, and purified using the Adeno-X-Maxi Purification kit (Clontech).

KrasLA2/{alpha}1KO cells (4 x 105 per 10-cm dishes) were incubated with serum-free medium containing 4 x 107 plaque-forming unit of AdVo or Ad{alpha}1. After 4 h, complete medium was added to the cells. After 2 d, the cells were transduced again as indicated above with a total of 3 independent treatments. Pools of KrasLA2/{alpha}1KO cells expressing high levels of the full-length integrin {alpha}1 subunit (KrasLA2/{alpha}1KO-Ad{alpha}1) were selected by flow cytometry using an antibody to the extracellular domain (Calbiochem). The expression of membrane-associated human integrin {alpha}1 in the sorted cell populations was stable for at least 7 d, allowing analysis of cell adhesion, morphology, anchorage-independent, and collagen-dependent growth.

Immunofluorescence. Primary lung tumor cells isolated from KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice were plated in complete medium on chamber slides. After 2 d, the cells were fixed in 4% formaldehyde and permeabilized with 0.1% Triton X-100 in PBS for 5 min. After blocking with 3% bovine serum albumin (BSA) in PBS, cells were incubated with anti-mouse ZO-1 (1:100; Molecular Probes, Inc.), anti-pan cytokeratin (1:100; DAKO), or anti-{alpha} smooth muscle actin (1:100; Sigma) antibodies followed by anti FITC-conjugated secondary antibodies (Calbiochem). Slides were mounted with anti-fade mounting medium (Vectashield; Vector Laboratories) and analyzed under an epifluorescence microscope (Nikon).

To analyze cell morphology, tumor cells were plated in serum-free medium on chamber slides coated with 2.5 µg/mL collagen IV (a specific integrin {alpha}1β1 ligand) or 2.5 µg/mL fibronectin (an integrin {alpha}1β1–independent ligand, used as positive control; all from Sigma). After 1 h, the cells were fixed, permeabilized, and stained with rhodamine-phalloidin (Molecular Probes). Cells were subsequently washed with PBS and examined under a fluorescence microscope (Nikon). Three independent experiments were performed.

Cell adhesion. Cell adhesion assay was performed as previously described (26). Briefly, 96-well plates were coated with different doses of fibronectin or collagen IV for 1 h at 37°C. After blocking nonspecific adhesion with 1% BSA in PBS, 5 x 104 tumor cells in 100 µL serum-free DMEM were added to the plates and incubated for 1 h at 37°C. The adherent cells were fixed with 4% formaldehyde, stained with 1% crystal violet, solubilized in 20% acetic acid, and the absorbance read at 595 nm. Cell adhesion to 1% BSA-coated wells was subtracted from the values obtained on extracellular matrix proteins. Three independent experiments were performed in quadruplicates.

Cell proliferation. Lung tumor cell proliferation was evaluated as described (27). Lung tumor cells (5 x 103 per well) were plated in low serum (1% FCS) onto 96-well plates coated with 10 µg/mL collagen IV. After 6 h, the cells were gently washed and incubated with serum-free medium with or without various concentrations (0–30 µmol/L final) of the MAP/ERK kinase (MEK)1/MEK2 inhibitors UO126 or PD98059, or the p38 MAPK inhibitors SB202190 or PD169316 (all from Calbiochem) in the presence of [3H]thymidine (0.5 µCi per well). After 48 h, the cells were collected and the amount of incorporated [3H]thymidine was analyzed as previously described (27). Four independent experiments were performed in quadruplicates.

Western blot analysis. Serum-starved tumor cells (3 x 104) were embedded for 0, 5, and 15 min in 30 µL collagen I+IV gels composed of 1 mg/mL rat tail collagen I, 30 µg/mL collagen IV, and DMEM containing 20 mmol/L HEPES (pH 7.2). Equal volumes of Laemmli buffer containing β-mercaptoethanol were subsequently added to the gels. Samples were then sonicated, boiled, and ran on a 10% SDS-PAGE gel and subsequently transferred to nitrocellulose membranes. Membranes were incubated with anti–phospho-ERK, anti–phospho-p38 MAPK, anti–phospho-Akt, or anti-Akt, anti-ERK, and anti-p38 MAPK antibodies (all from Cell Signaling) followed by the appropriate HRP-conjugated secondary antibodies. Immunoreactive bands were identified using enhanced chemiluminescence according to the manufacturer's instructions. Four independent experiments were performed.

To determine activated ERK and p38 MAPK in vivo, tumors derived from 120-d-old KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice were pulverized in liquid nitrogen and suspended in radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.4), 1% Triton-X100, 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, proteinease inhibitor-cocktail 1 mmol/L Na3VO4, and 1 mmol/L NaF]. Equal amount of total tumor proteins (30 µg/lane) were loaded on a 10% SDS-PAGE and ran under reducing conditions. Membranes were subsequently incubated with anti–phospho-ERK, anti–phospho-p38, anti-ERK, and anti-p38 antibodies as indicated above.

Soft agar colony formation assay. Lung tumor cells (2.5 x 104) were suspended in equal volumes of 1.2% agar and 2x DMEM/20% FCS, and plated onto 24-well plate coated with agar. Normal medium supplemented with 10% fetal bovine serum was added to the top of the gelled matrix. After 7 to 8 d, colonies were counted in 5 random fields per well and photographed. Three independent experiments were performed in triplicates.

In vivo tumorigenicity. To determine the ability of primary lung tumor cells to form tumors in vivo, nude mice (9 mice per cell type) were injected s.c. with either 2.5 x 105 or 1 x 106 tumor cells in 200 µL PBS. Tumor growth was then analyzed over time. Five weeks posttumor injection, mice were sacrificed and the number and size of tumors were evaluated. Tumor volumes were calculated using the following formula: tumor volume (mm3) = (length x width2)/2 (12). All animal protocols were reviewed and approved by Vanderbilt University Medical Center Institutional Animal Care and Use Committee.

Statistical analysis. The Student's t test was used for comparisons between two groups, and ANOVA using Sigma-Stat software for statistical difference between multiple groups. A P value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Increased survival and decreased incidence of lung cancer in KrasLA2/{alpha}1KO mice. To investigate the role of integrin {alpha}1β1 in the control of primary lung tumor development, we crossed WT and {alpha}1KO mice with KrasLA2 mice (25). As shown in Fig. 1A , KrasLA2/{alpha}1KO mice showed significantly increased survival rates compared with KrasLA2/{alpha}1WT (336 ± 119 days versus 279 ± 85 days; P = 0.03). To determine the reason for this difference in survival, mice were sacrificed at different time points and incidence, number, and size of lung tumors evaluated. As shown in Fig. 1B, lung tumors developed in both KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice (tumors from 120-day-old mice shown); however, the incidence was significantly reduced in the latter group [95% in KrasLA2/{alpha}1WT mice (n = 36) versus 75% in KrasLA2/{alpha}1KO mice (n = 24); P < 0.05]. In addition, the number of visible tumors on the lung surface (Fig. 1B and C) and their size (Fig. 1D) was significantly reduced in the KrasLA2/{alpha}1KO mice.


Figure 1
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  		Figure 1. Increased survival and reduced tumor development in KrasLA2/{alpha}1KO mice. A, KrasLA2 mice crossed with the {alpha}1KO mice (KrasLA2/{alpha}1KO) showed significantly increased survival compared with KrasLA2 mice crossed with integrin {alpha}1 WT mice (KrasLA2/{alpha}1WT) with a mean age of death per sacrifice of 279 ± 85 d for the KrasLA2/{alpha}1WT versus 336 ± 95 d for the KrasLA2/{alpha}1KO mice (P = 0.03). B, photograph of the lungs of KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO male mice sacrificed 120 d after birth (top). Scale bar, 5 mm. Bottom, H&E staining of lungs of KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice. Magnification, x200. Scale bar in the inset, 20 µm. C and D, KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice were sacrificed 120 d after birth and tumor number (C) and size (D) were evaluated. The number of tumors visible on the lung surface was evaluated and expressed as average number of tumors per lung (C). Tumor diameter was measured with a caliper in 14 KrasLA2/{alpha}1WT and 102 tumors from 24 KrasLA2/{alpha}1KO mice, and tumors were divided into three groups as indicated (D).

 
Decreased vascularization and proliferation in lung tumors from KrasLA2/{alpha}1KO mice. As loss of integrin {alpha}1β1 leads to decreased tumor growth due to reduced tumor-associated vasculature (9, 10, 12, 13), tumor sections were stained with anti-CD31 antibody. Tumors derived from the KrasLA2/{alpha}1KO mice not only were significantly less vascularized (Fig. 2A and B ) but also showed reduced tumor cell proliferation (Fig. 2A and B) and increased apoptosis (Fig. 2A and B) compared with tumors from KrasLA2/{alpha}1WT mice.


Figure 2
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  		Figure 2. Reduced tumor growth, vascularization, and ERK activation in lung tumors from KrasLA2/{alpha}1KO mice. A, paraffin sections of lung tumors from 120-d-old KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice were stained with anti-CD31 or anti-PCNA antibodies or subjected to Dead End colorimetric TUNEL system for the evaluation of vascularization, proliferation, and apoptosis, respectively. B, quantification of area occupied by CD31-positive structures per microscopic field, as well as the number of PCNA or TUNEL-positive cells per microscopic field was evaluated as described in the Materials and Methods. Columns, mean of 90 image per genotype; bars, SD. *, significant differences (P < 0.05) between the two genotypes. C, paraffin lung sections were stained with anti–phospho-ERK and anti–phospho-p38 MAPK antibodies for the evaluation of activated ERK and p38 MAPK, respectively. The percentage of anti–phospho-ERK and anti–phospho-p38 MAPK–positive cells was evaluated as described in the Materials and Methods. Values and * are as in A. D, total tumor lysates (30 µg per lane) were prepared from tumors isolated from 120-d-old mice as described in the Materials and Methods, and Western blot analysis was performed using anti–phospho-ERK, anti–phospho-p38 MAPK, anti-ERK, and anti-p38 MAPK–specific antibodies. Three independent experiments with a total of 8 KrasLA2/{alpha}1WT (w) and 9 KrasLA2/{alpha}1KO (k) mice are shown.

 
Although decreased vascularization might account for the smaller lung tumors in KrasLA2/{alpha}1KO mice, loss of integrin {alpha}1β1 expression by the tumor cells could contribute to the decreased proliferation and increased apoptosis, as integrin {alpha}1β1 promotes cell survival and proliferation on collagenous substrata via activation of the Shc/Grb2/ERK pathway (7) and inhibition of the proapoptotic p38 MAPK (28). When activated ERK and p38 MAPK kinases were determined in tumor sections, a significant reduction in the levels of phospho-ERK but an increase in phospho-p38 MAPK was detected in tumors from KrasLA2/{alpha}1KO mice (Fig. 2C and D). Thus, decreased activation of promitogenic molecules together with decreased vascularization contributes to the decreased tumor size in the KrasLA2/{alpha}1KO mice.

KrasLA2/{alpha}1KO primary lung epithelial tumor cells fail to adhere and spread on collagen IV. Host-tumor interactions plays a key role in cancer progression, and the recruitment of endothelial cells and leukocytes facilitate both tumor growth and progression (reviewed by ref. 29). As loss of integrin {alpha}1β1 leads to decreased tumor-associated angiogenesis (9, 10) and leukocyte recruitment to the site of injury (30, 31), we sought to determine whether the decreased number, size, and proliferation rate of tumors from KrasLA2/{alpha}1KO mice could be attributed to the loss of this receptor by the tumor cells themselves. For this reason, we derived primary cultures of epithelial cells from lung tumors isolated from KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice. The epithelial nature of the cells was confirmed by performing staining with both epithelial and nonepithelial markers. Both cell types showed an epithelial morphology (Supplementary Fig. S1A), stained positive for the epithelial markers ZO-1 and cytokeratins (Supplementary Fig. S1B), and did not express {alpha}-smooth muscle actin (Supplementary Fig. S1B).

The ability of the tumor cells to adhere to and spread on collagen IV, a major integrin {alpha}1β1 ligand highly expressed in the basement membranes of the lungs (32), was determined. In contrast to KrasLA2/{alpha}1WT cells, the KrasLA2/{alpha}1KO tumor cells showed impaired adhesion and spreading on collagen IV (Fig. 3A and B ) but not on fibronectin (Fig. 3A and B), an integrin {alpha}1β1-independent ligand. To determine whether the adhesion and spreading defect of KrasLA2/{alpha}1KO tumor cells on collagen IV was a direct consequence of loss of {alpha}1β1, we transduced KrasLA2/{alpha}1KO tumor cells with an AdVo or Ad{alpha}1 to generate KrasLA2/{alpha}1KO-AdVo and KrasLA2/{alpha}1KO-Ad{alpha}1–expressing cells, respectively (Supplementary Fig. S1C). As shown in Fig. 3A and B, KrasLA2/{alpha}1KO-Ad{alpha}1, but not KrasLA2/{alpha}1KO-AdVo cells, adhered and spread on collagen IV to a similar degree as KrasLA2/{alpha}1WT cells. These findings not only show that the decreased adhesion and spreading on collagen IV in the KrasLA2/{alpha}1KO cells is a direct consequence of the loss of this receptor but also verify that the properties of the KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO-Ad{alpha}1 cells are the same with respect to integrin {alpha}1β1-dependent functions.


Figure 3
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  		Figure 3. Lung epithelial tumors cells from KrasLA2/{alpha}1KO mice show reduced adhesion and spreading on collagen IV. A, the tumor cells indicated (5 x 104) were plated onto 96-well plates coated with collagen IV (left) or fibronectin (right) at the concentrations indicated for 1 h in serum-free medium. Adherent cells were then stained with crystal violet, lysed, and the absorbance measured. Points, mean of one representative experiment performed in quadruplicate; bars, SD. *, significant differences (P < 0.05) between KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO cells; **, significant differences (P < 0.05) between KrasLA2/{alpha}1K0-Ad{alpha}1 and KrasLA2/{alpha}1KO-AdVo cells. B, the tumor cells indicated were plated on 4 µg/mL collagen IV or fibronectin in serum-free medium. After 1 h, the cells were fixed and stained with rhodamine-phalloidin. Bar, 10 µm.

 
Reduced anchorage-independent and anchorage-dependent growth in KrasLA2/{alpha}1KO primary lung epithelial tumor cells. We next determined anchorage-independent growth of KrasLA2/{alpha}1WT, KrasLA2/{alpha}1KO, KrasLA2/{alpha}1KO-AdVo, and KrasLA2/{alpha}1KO-Ad{alpha}1 lung epithelial tumor cells by evaluating colony formation 7 to 8 days after plating on soft agar. A significant reduction in the ability to form colonies was observed in the KrasLA2/{alpha}1KO and KrasLA2/{alpha}1KO-AdVo cells compared with their KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO-Ad{alpha}1 counterparts (Fig. 4A and B ), suggesting a role for integrin {alpha}1β1 in anchorage-independent growth. Similarly, when cells were grown on collagen IV, thus primarily allowing the engagement of collagen binding integrins, only KrasLA2/{alpha}1WT and KRasLA2/{alpha}1KO-Ad{alpha}1 tumor cells showed sustained proliferation (Fig. 4C).


Figure 4
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  		Figure 4. Decreased anchorage-independent, anchorage-dependent, and in vivo growth of KrasLA2/{alpha}1KO tumor epithelial cells. A and B, the lung epithelial tumor cells indicated were plated onto soft agar as described in the Materials and Methods, and colony number was evaluated 7 to 8 d after plating. Columns, mean of one representative experiment performed in triplicate (five fields per well were analyzed); bars, SD. C, the lung epithelial tumor cells indicated were plated in 96-well plates coated with 10 µg/mL collagen IV. Six hours later, the cells were incubated with serum-free medium containing 3H-Thymidine (0.5 µCi per well) for further 48 h, and proliferation was then evaluated as described in the Materials and Methods. Columns, mean of one representative experiment performed in quadruplicate; bars, SD. Cpm, counts per minute. D, 2.5 x 105 or 1 x 106 KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO tumor cells were injected s.c. into nude mice (n = 9), and tumor number and volume were analyzed 5 wk postinjection. Circles, individual tumor volume; bar, mean.

 
To determine whether the differences in growth observed in vitro were reflected in vivo, the tumorigenic activity of KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO tumor cell populations was tested by injecting nude mice s.c. with various amounts of tumor cells. These assays were not performed with the KRasLA2/{alpha}1KO-Ad{alpha}1 tumor cells as they only retain integrin {alpha}1 expression for ~7 days. As shown in Fig. 4D, KrasLa2/{alpha}1WT cells form visible tumors in all mice within 5 weeks of injection and their tumorigenic potential was independent of the number of cells injected. In contrast, KrasLA2/{alpha}1KO cells formed few small visible tumors only when injected in high numbers. Taken together these, results confirm our finding that expression of integrin {alpha}1β1 plays a key role in the formation and growth of KrasLA2 lung tumors.

Reduced ERK activation accounts for decreased proliferation of KrasLA2/{alpha}1KO primary lung epithelial tumor cells. The ability of the tumor cells to activate ERK, p38 MAPK, and Akt/PKB upon binding to collagen was determined as (a) oncogenic Ras supports cell survival and proliferation by activating ERK (33) and down-regulating p38 MAPK activation (34, 35); (b) decreased ERK but increased p38 MAPK activation was observed in tumors derived from KrasLA2/{alpha}1KO mice (Fig. 2C and D); (c) activation of integrin {alpha}1β1 results in increased ERK phosphorylation via the Shc/Grb2 pathway (7); and (d) Kras promotes growth transformation by activation of the phosphatidylinositol-3-OH kinase (PI3K) pathway (36). These experiments were performed on KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO tumor cells only as KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO-Ad{alpha}1 behave in a similar fashion (see data above). As shown in Fig. 5A , increased baseline activation of ERK but similar levels of phosphorylated Akt and p38 MAPK were evident in serum-starved KrasLA2/{alpha}1WT when compared with KrasLA2/{alpha}1KO tumor cells. Interestingly, when embedded within collagen gels, sustained ERK activation was only observed in KrasLA2/{alpha}1WT tumor cells, whereas it decreased significantly in KrasLA2/{alpha}1KO tumor cells within 15 minutes from plating. In contrast, p38 MAPK activation decreased over time in KrasLA2/{alpha}1WT tumor cells plated in collagen gels, and it remained activated KrasLA2/{alpha}1KO tumor cells (Fig. 5A). Finally, comparable levels of collagen-mediated Akt activation were observed in both KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO tumor cells (Fig. 5A). Thus, sustained ERK activation and down-regulation of p38 MAPK phosphorylation in the KrasLA2/{alpha}1WT tumor cells exposed to a collagenous environment requires the expression of integrin {alpha}1β1, suggesting that oncogenic Kras per se is not sufficient to regulate the activation state of these kinases.


Figure 5
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  		Figure 5. ERK mediates KrasLA2/{alpha}1WT tumor epithelial cell growth on collagen substrata. A, equal number of serum-starved lung epithelial tumor cells was embedded in collagen I+IV gels for the time indicated (min). The gels were subsequently sonicated, subjected to SDS-PAGE, and levels of phosphorylated ERK, p38 MAKP, and Akt as well as total ERK, p38 MAKP, and Akt were detected by immunoblotting. Images are representative of three independent experiments. B, tumor cells were subjected to proliferation assay as described in Fig. 4 in the presence or absence of the kinase inhibitors indicated. Columns, mean of one representative experiment performed in quadruplicate; bars, SD. *, statistically significant differences (P < 0.05) between KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO cells; **, statistically significant differences (P < 0.05) between untreated and inhibitor-treated KrasLA2/{alpha}1WT cells.

 
To determine the contribution of ERK in KrasLA2/{alpha}1WT tumor cell proliferation, tumor cells were embedded within collagen I+IV gels with or without various concentrations of the MEK inhibitors UO126 or PD98059. KrasLA2/{alpha}1WT tumor cells incubated with the ERK inhibitors showed a marked dose-dependent reduction in cell proliferation (Fig. 5B). Importantly, these inhibitors had no measurable effect on the proliferation of KrasLA2/{alpha}1KO tumor cells, demonstrating their effects are not due to cell toxicity. As expected from the signaling data, the use of the p38 MAPK inhibitors, PD169316 or SB203580, did not have any effect on the proliferation of KrasLA2/{alpha}1WT or KrasLA2/{alpha}1KO tumor cells, strongly supporting a role for ERK but not p38 MAPK in sustaining proliferation of KrasLA2/{alpha}1WT lung epithelial tumor cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
The contribution of integrin {alpha}1β1 in tumor progression has only been investigated with respect to tumor-associated angiogenesis in xenograft and orthotopic models of cancer (9, 10, 12, 13). These studies indicate that lack of integrin {alpha}1β1 expression by endothelial cells protects the host from tumor growth by reducing pathologic angiogenesis. As it is unknown whether deleting the integrin {alpha}1 subunit also influences tumor initiation and formation, we crossed {alpha}1KO mice with KrasLA2 mice, a model for spontaneous NSCLC (25). We show that the KrasLA2/{alpha}1KO mice had a decreased incidence, number, and size of primary lung adenomas resulting in a longer life span than KrasLA2/{alpha}1WT mice. Primary tumor cells derived from KrasLA2/{alpha}1KO mice had decreased tumorigenicity when injected into nude mice, formed fewer anchorage-independent colonies, and proliferated less than KrasLA2/{alpha}1WT cells on collagen. These results show that integrin {alpha}1β1 plays a key role in both the development and growth of lung cancer, which is independent of integrin {alpha}1β1-mediated angiogenesis.

The KrasLA2 mouse model allowed us to show for the first time that the collagen-binding receptor, integrin {alpha}1β1, plays a critical role in the initiation and development of lung tumors in their natural environment. Although a limitation of the KrasLA2 model is that the tumors rarely metastasize, it has the great advantage that it does not rely on the exogenous injection of tumor cell lines.

Our in vivo analysis clearly indicates that lack of integrin {alpha}1 in the KrasLA2 background results in a decreased incidence and number of lung tumors and prolonged life span of the mice. A likely explanation for this is that that integrin {alpha}1β1 supports cell survival and proliferation on collagens substrata as previously shown (7, 11); collagen IV is one of the major extracellular matrix in the lungs (32); and integrin activation is required for collagen IV–mediated proliferation of human lung cancer cells (37). The finding that lung tumors isolated from the KrasLA2/{alpha}1KO mice were also less vascularized than those derived from KrasLA2/{alpha}1WT mice supports our previous finding that genetic ablation of integrin {alpha}1β1 results in decreased tumor-associated angiogenesis (9, 10, 12, 13), and integrin {alpha}1–blocking antibodies prevent growth factor–mediated angiogenesis in a corneal assay model (38).

Comparing primary lung epithelial tumor cells from both KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice allowed us to show that integrin {alpha}1β1 expression by the tumor cells played a critical role in the increased tumorigenicity seen in vivo. Although oncogenic Kras is known to promote anchorage-independent growth of human pancreatic cells via Raf and PI3K activation (36), we show that expression of integrin {alpha}1β1 increases the ability of KrasLA2 lung tumor cells to undergo anchorage-independent growth, as well as in vivo tumor formation. This result agrees with the finding that oncogenic changes in the Kras gene alone are insufficient to confer a malignant phenotype to human bronchial epithelial cells (39). Moreover, similar to our KrasLA2/{alpha}1KO cells, down-regulation of the integrin β4 subunit in tumor cells results in decreased anchorage-independent growth (40). Similarly, it has been recently shown that a subpopulation of MCF-7 cells that are more tumorigenic in vivo and more capable of anchorage-independent growth than their parental line are characterized by overexpression of the integrin {alpha}6 subunit (41). Thus, it is conceivable that integrin {alpha}1β1, together with oncogenic Kras, is responsible for regulating and supporting anchorage-independent growth of lung tumor epithelial cells.

Our in vitro signaling studies show that within collagenous environments sustained ERK but decreased p38 MAPK activation of KrasLA2 lung tumor cells requires the presence of integrin {alpha}1β1. The regulation of ERK activation in Kras-transformed cells seems to be cell type dependent, as in endometrial cancer ERK activation occurs independently of oncogenic Kras (42), in Kras-transformed pancreatic tumor cells, ERK activation is down-regulated due to up-regulation of MAPK kinase phosphatase-2 (43, 44), and in human colon cancer cells stimuli such as radiation are sufficient to stimulate ERK signaling and cell cycle progression (45). Similar to ERK, the contribution of activated p38 MAPK to Kras-induced tumorigenesis is highly dependent on the cell type, as in Kras-transformed human colon tumor cell lines p38 MAPK activation is associated with increased apoptosis (46), whereas in Kras-transformed pancreatic carcinoma p38 MAPK activation is required for invasion (47). Therefore, in Kras-transformed lung tumor cells, expression of integrin {alpha}1β1 might cooperate with and/or act independently of oncogenic Kras to induce ERK but decrease p38 MAPK activation. This hypothesis is supported by our previous finding that integrin {alpha}1β1 is the only collagen receptor able to support cell survival and proliferation via activation of the Shc/Grb2/ERK pathway (7), whereas its loss results in increased p38 MAPK activation (28).

Our in vitro studies indicate that loss of integrin {alpha}1β1 results in decreased anchorage-independent and collagen-mediated growth, as well as adhesion and spreading on collagenous substrata. However, loss of this receptor in vivo seems to affect only cell proliferation and survival, as no overall differences in tissue architecture were observed between lung tumors from KrasLA2/{alpha}1WT and KrasLA2/{alpha}1KO mice. This result agrees with our previous finding that although primary {alpha}1-null endothelial cells fail to adhere and proliferate on collagenous substrata, no overall differences in vascular integrity are observed between WT and integrin {alpha}1KO mice at baseline (9, 10).

In conclusion, we provide novel evidence that loss of the integrin {alpha}1 subunit decreases the incidence and growth of lung adenomas initiated by oncogenic Kras. As expression of integrin {alpha}1β1 has been detected in many types of human cancers, including a subset of patients with non–small cell lung cancer (17), it is conceivable that Kras and integrin {alpha}1β1 cooperate to drive the growth of non–small cell lung cancer in humans.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: National Cancer Institute/NIH R01 CA94849-01 (A. Pozzi), RO1-DK 69921 and R01-DK075594 (R. Zent), a Merit award from the Department of Veterans Affairs (R. Zent), and NIH/Lung Specialized Programs of Research Excellence/P50CA090949 (D. Carbone).

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.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 4/14/08. Revised 5/13/08. Accepted 6/ 3/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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