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. Author manuscript; available in PMC: 2010 Jan 3.
Published in final edited form as: Cancer Res. 2008 Aug 1;68(15):6127–6135. doi: 10.1158/0008-5472.CAN-08-1395

Loss of integrin α1β1 ameliorates Kras-induced lung cancer

Ines Macias-Perez 1,2, Corina Borza 1, Xiwu Chen 1, Xuexian Yan 1, Raquel Ibanez 1, Glenda Mernaugh 1, Lynn M Matrisian 2, Roy Zent 1,2,3,4, Ambra Pozzi 1,2,4
PMCID: PMC2801028  NIHMSID: NIHMS162735  PMID: 18676835

Abstract

The collagen IV binding receptor integrin α1β1 has been shown to regulate lung cancer due to its pro-angiogenic properties, however it is unclear whether this receptor also plays a direct role in promoting primary lung tumors. To investigate this possibility, integrin α1-null mice were crossed with KrasLA2 mice which 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/α1-null mice have a decreased incidence of primary lung tumors and longer survival compared to KrasLA2/α1 wild type controls. Tumors from KrasLA2/α1-null mice were also smaller; less vascularized and exhibited reduced cell proliferation and increased apoptosis, as determined by PCNA and TUNEL staining, respectively. Moreover, tumors from the KrasLA2/α1-null mice showed diminished ERK, but enhanced p38 MAPK activation. Primary lung tumor epithelial cells isolated from KrasLA2/α1-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 to KrasLA2/α1 wild type tumor cells. These results indicate that loss of the integrin α1 subunit decreases the incidence and growth of lung epithelial tumors initiated by oncogenic Kras, suggesting that both Kras and integrin α1β1 cooperate to drive the growth of non-small cell lung cancer in vivo.

Keywords: oncogenes, receptors, lung, cancer, initiation

INTRODUCTION

Integrins are extracellular matrix receptors composed of non-covalently associated α 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 four major collagen receptors, namely integrins α1β1, α2β1, α10β1, and α11β1 (3-6). Integrin α1β1 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 (MMP) (8-10) and reactive oxygen species (11).

The contribution of integrin α1β1 in tumor formation and progression is poorly defined. Using xenograft and orthotopic models of cancer, we showed that integrin α1-null mice develop less, smaller, and poorly vascularized tumors compared to wild type mice. The reduced angiogenesis in the α1-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 α1β1 serves as a pro-angiogenic receptor, and loss of its expression by endothelial cells protects the host from tumor growth by reducing pathological angiogenesis.

It is unclear whether integrin α1β1 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 α1β1 in melanoma can either correlate with enhanced progression/metastasis (14, 15) as well with a favorable outcome (16). Integrin α1β1 has also been implicated in pulmonary neoplasms and its expression is upregulated in bronchoalveolar carcinomas and pulmonary carcinoids (17). In contrast, down-regulation of this receptor was observed in malignant breast tumors (18) while 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 α1β1 in regulating tumor cell adhesion, migration and invasion have been documented in vitro. For example, integrin α1β1 contributes to tumor cell invasion by inducing MMP3 expression in mouse mammary carcinoma cells (20), while 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 αvβ3- and α6β4-mediated Ras activation is necessary to support cell proliferation (22, 23). Integrin α1β1 is linked to Ras signaling via the adaptor protein Shc (24) and supports cell survival via the Shc/Grb2/ERK axis (7). Given that Ras family members are proto-oncogenes and integrin α1β1 can activate the RAS-MAPK pathway (7, 24), it is conceivable that α1β1 cooperates with the Ras signaling in regulating tumorigenesis. To test this hypothesis, integrin α1-null mice were crossed to the KrasLA2 mouse, a model of spontaneous non-small cell lung cancer. 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 demonstrate that the KrasLA2/α1-null mice have a decreased incidence, number and size of primary lung tumors resulting in longer survival than KrasLA2/α1 wild type controls. Moreover, primary lung tumor epithelial cells isolated from KrasLA2/α1-null mice show decreased anchorage-independent colony formation, collagen-mediated cell growth, and, most importantly, tumorigenicity when injected into nude mice compared to KrasLA2/α1 wild type tumor cells. Thus, integrin α1β1 plays a key role in both the development and growth of lung cancer that is independent of integrin α1β1-mediated angiogenesis. Furthermore, these data suggest that this collagen receptor and oncogenic Kras cooperate to drive lung tumorigenesis.

MATERIALS AND METHODS

The KrasLA mouse model of lung cancer

KrasLA2 mice (gift of Dr. T. Jacks, MIT) (25) were crossed with wild type (WT) or integrin α1-null (α1KO) mice on the SV129 background [generated as described in (6)] to obtain KrasLA2/α1WT and KrasLA2/α1KO mice. Since the K-rasLA allele is non-functional in the germline configuration (25), the mice were maintained in a heterozygous state, as described (25). Age- and sex-matched KrasLA2/α1WT and KrasLA2/α1KO were sacrificed at different time points ranging from 7-250 days of age as these mice develop multiple lung tumors as early as one week after birth and do not usually survive more than 200 days (25). Representative analysis of KrasLA2/α1WT and KrasLA2/α1KO mice sacrificed 120 days after birth are shown. The lungs were removed immediately at sacrifice and the number of tumors visible on the lung surface counted (36 KrasLA2/α1WT and 24 KrasLA2/α1KO mice were analyzed). Tumor diameter was measured with a caliper and tumors were divided into three groups: 0-2 mm, 2-5 mm and >5 mm tumor diameter (170 tumors from 14 KrasLA2/α1WT and 102 tumors from 24 KrasLA2/α1KO mice were used for analysis). Parts of lungs containing tumors were fixed in 4% paraformaldehyde and processed for histological analysis or immunohistochemistry.

Survival analysis

KrasLA2/α1WT and KrasLA2/α1KO mice were analyzed daily from birth and sacrificed at the first sign of shortness of breath, reduced locomotion and reduced body weight (greater than 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. 25 mice/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 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 horseradish peroxidase–conjugated secondary antibodies (1:200, Jackson) and Sigma Fast DAB chromogenic tablets (Sigma). CD31 positive structures within tumors were recorded (3 images/tumor with 4 tumors/lung with a total of 10 lungs/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 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/tumor with 4 tumors/lung with a total of 10 lungs/genotype) per microscopic field (400×).

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/α1WT and KrasLA2/α1KO mice

Age-matched KrasLA2/α1WT and KrasLA2/α1KO male mice were sacrificed and the lungs were perfused with PBS. Lungs were then removed, tumors excised, minced and incubated with 0.25% trypsin/1 mM EDTA for 10 minutes 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 minutes at 1000 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 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 tumors cells were performed. Each preparation was derived from tumors pooled from 6-9 KrasLA2/α1WT and 8-10 KrasLA2/α1KO 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 α1 subunit into KrasLA2/α1KO cells

The human integrin α1 cDNA (gift of Dr. E. Marcantonio) was subcloned into pAdeno-X (Clontech) following manufacturer instructions. Empty adenovirus (AdVo) and adenovirus expressing the integrin α1 subunit (Adα1) were transfected in HEK cells, amplified, and purified using the Adeno-X-Maxi Purification kit (Clontech).

KrasLA2/α1KO cells (4×105/10 cm dishes) were incubated with serum-free medium containing 4×107 pfu of AdVo or Adα1. After 4 hours, complete medium was added to the cells. After 2 days the cells were transduced again as indicated above with a total of three independent treatments. Pools of KrasLA2/α1KO cells expressing high levels of the full-length integrin α1 subunit (KrasLA2/α1KO-Adα1) were selected by flow cytometry using an antibody to the extracellular domain (Calbiochem). The expression of membrane-associated human integrin α1 in the sorted cell populations was stable for at least 7 days, allowing analysis of cell adhesion, morphology, anchorage-independent and collagen-dependent growth.

Immunofluorescence

Primary lung tumor cells isolated from KrasLA2/α1WT and KrasLA2/α1KO mice were plated in complete medium on chamber slides. After 2 days the cells were fixed in 4% formaldehyde and permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. After blocking with 3% BSA in PBS, cells were incubated with anti-mouse ZO-1 (1:100, Molecular Probes Inc), anti-pan cytokeratin (1:100, DAKO), or anti-α 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 Labs.) 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 α1β1 ligand) or 2.5 μg/ml fibronectin (an integrin α1β1 independent ligand, used as positive control) (all from Sigma). After 1 hour 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 hour at 37°C. After blocking nonspecific adhesion with 1% BSA in PBS, 5×104 tumor cells in 100 μl serum-free DMEM were added to the plates and incubated for 1 hour 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 ECM proteins. Three independent experiments were performed in quadruplicates.

Cell proliferation

Lung tumor cell proliferation was evaluated as described (27). Lung tumor cells (5×103/well) were plated in low serum (1% FCS) onto 96-well plates coated with 10 μg/ml collagen IV. After 6 hours, the cells were gently washed and incubated with serum-free medium with or without various concentrations (0-30 μM final) of the MEK1/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/well). After 48 hours, the cells were collected and the amount of incorporated [3H]thymidine analyzed as previously described (27). Four independent experiments were performed in quadruplicates.

Western Blot Analysis

3×104 serum-starved tumor cells were embedded for 0, 5 and 15 minutes 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 mM 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 day old KrasLA2/α1WT and KrasLA2/α1KO mice were pulverized in liquid nitrogen and suspended in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Triton-X100, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, proteinease inhibitor-cocktail 1 mM Na3VO4, 1 mM 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

2.5 × 105 lung tumor cells were suspended in equal volumes of 1.2% agar and 2× DMEM/20% FCS and plated onto 24-well plate coated with agar. Normal media supplemented with 10% FBS was added to the top of the gelled matrix. After 7-8 days, colonies were counted in five 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/cell type) were injected subcutaneously with either 2,5×105 or 1×106 tumor cells in 200 μl PBS. Tumor growth was then analyzed over time. Five weeks post tumor 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 × 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 analysis of variance using Sigma-Stat software for statistical difference between multiple groups. p<0.05 was considered statistically significant.

RESULTS

Increased survival and decreased incidence of lung cancer in KrasLA2/α1-null mice

To investigate the role of integrin α1β1 in the control of primary lung tumor development we crossed wild type (WT) and integrin α1-null (α1KO) mice with KrasLA2 mice (25). As shown in Figure 1A, KrasLA2/α1KO mice showed significantly increased survival rates compared to KrasLA2/α1WT (336+/−119 days vs. 279+/−85 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 Figure 1B, lung tumors developed in both KrasLA2/α1WT and KrasLA2/α1KO mice (tumors from 120 day old mice shown), however the incidence was significantly reduced in the latter group [95% in KrasLA2/α1WT mice (n=36) vs. 75% in KrasLA2/α1KO mice (n=24), p<0.05]. In addition, the number of visible tumors on the lung surface (Figs. 1B and 1C) and their size (Fig. 1D) was significantly reduced in the KrasLA2/α1KO mice.

Figure 1. Increased survival and reduced tumor development in KrasLA2/α1KO mice.

Figure 1

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(A) KrasLA2 mice crossed with the integrin α1-null mice (KrasLA2/α1KO) showed significantly increased survival compared to KrasLA2 mice crossed with integrin α1 wild type mice (KrasLA2/α1WT) with a mean age of death/sacrifice of 279 ± 85 days for the KrasLA2/α1WT vs. 336 ± 95 days for the KrasLA2/α1KO mice (p=0.03). (B) Upper panel: Photograph of the lungs of KrasLA2/α1WT and KrasLA2/α1KO male mice sacrificed 120 days after birth. Scale bar, 5 mm. Lower panel: Hematoxylin and eosin staining of lungs of KrasLA2/α1WT and KrasLA2/α1KO mice. Magnification, 200×. Scale bar in the inset, 20 μm. (C, D) KrasLA2/α1WT and KrasLA2/α1KO mice were sacrificed 120 days 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/α1WT and 102 tumors from 24 KrasLA2/α1KO mice and tumors were divided into three groups as indicated (D).

Decreased vascularization and proliferation in lung tumors from KrasLA2/α1KO mice

As loss of integrin α1β1 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/α1KO mice not only were significantly less vascularized (Figs. 2A and 2B), but also showed reduced tumor cell proliferation (Figs. 2A and 2B) and increased apoptosis (Figs. 2A and 2B) compared to tumors from KrasLA2/α1WT mice.

Figure 2. Reduced tumor growth, vascularization, and ERK activation in lung tumors from KrasLA2/α1KO mice.

Figure 2

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(A) Paraffin sections of lung tumors from 120 day old KrasLA2/α1WT and KrasLA2/α1KO 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. Values indicate the mean ± SD of 90 image/genotype. (*) indicates 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 Methods. Values and (*) are as in (A). (D) Total tumor lysates (30 μg/lane) were prepared from tumors isolated from 120 day old mice as described in the 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/α1WT (w) and 9 KrasLA2/α1KO (k) mice are shown.

Although decreased vascularization might account for the smaller lung tumors in KrasLA2/α1KO mice, loss of integrin α1β1 expression by the tumor cells could contribute to the decreased proliferation and increased apoptosis, as integrin α1β1 promotes cell survival and proliferation on collagenous substrata via activation of the Shc/Grb2/ERK pathway (7) and inhibition of the pro-apoptotic 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/α1KO mice (Figs. 2C and 2D). Thus, decreased activation of pro-mitogenic molecules together with decreased vascularization contributes to the decreased tumor size in the KrasLA2/α1KO mice.

KrasLA2/α1KO 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 (29)]. As loss of integrin α1β1 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/α1KO 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/α1WT and KrasLA2/α1KO mice. The epithelial nature of the cells was confirmed by performing staining with both epithelial and non-epithelial 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 α-smooth muscle actin (Supplementary Fig. S1B).

The ability of the tumor cells to adhere to and spread on collagen IV, a major integrin α1β1 ligand highly expressed in the basement membranes of the lungs (32), was determined. In contrast to KrasLA2/α1WT cells, the KrasLA2/α1KO tumor cells showed impaired adhesion and spreading on collagen IV (Figs. 3A and 3B) but not on fibronectin (Figs. 3A and 3B), an integrin α1β1-independent ligand. To determine whether the adhesion and spreading defect of KrasLA2/α1KO tumor cells on collagen IV was a direct consequence of loss of α1β1, we transduced KrasLA2/α1KO tumor cells with an empty adenovirus (AdVo) or adenovirus carrying the human integrin α1 cDNA (Adα1) in order to generate KrasLA2/α1KO-AdVo and KrasLA2/α1KO-Adα1 expressing cells, respectively (Supplementary Fig. S1C). As shown in Figures 3A and 3B, KrasLA2/α1KO-Adα1, but not KrasLA2/α1KO-AdVo cells, adhered and spread on collagen IV to a similar degree as KrasLA2/α1WT cells. These findings not only demonstrate that the decreased adhesion and spreading on collagen IV in the KrasLA2/α1KO cells is a direct consequence of the loss of this receptor, but also verify that the properties of the KrasLA2/α1WT and KrasLA2/α1KO-Adα1 cells are the same with respect to integrin α1β1-dependent functions.

Figure 3. Lung epithelial tumors cells from KrasLA2/α1KO mice show reduced adhesion and spreading on collagen IV.

Figure 3

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(A) The tumor cells indicated (5×104) were plated onto 96-well plates coated with collagen IV (left panel) or fibronectin (right panel) at the concentrations indicated for 1 hour in serum-free medium. Adherent cells were then stained with crystal violet, lysed, and the O.D. measured. Data represent the mean ± SD of one representative experiment performed in quadruplicate. (*) indicates significant differences (p<0.05) between KrasLA2/α1WT and KrasLA2/α1KO cells; while (**) indicates significant differences (p<0.05) between KrasLA2/α1K0-Adα1 and KrasLA2/α1KO-AdVo cells. (B) The tumor cells indicated were plated on 4 μg/ml collagen IV or fibronectin in serum free. After 1 hour, the cells were fixed and stained with rhodamine-phalloidin. Bar, 10 μm.

Reduced anchorage-independent and -dependent growth in KrasLA2/α1KO primary lung epithelial tumor cells

We next determined anchorage-independent growth of KrasLA2/α1WT, KrasLA2/α1KO, KrasLA2/α1KO-AdVo and KrasLA2/α1KO-Adα1 lung epithelial tumor cells by evaluating colony formation 7-8 days after plating on soft agar. A significant reduction in the ability to form colonies was observed in the KrasLA2/α1KO and KrasLA2/α1KO-AdVo cells compared to their KrasLA2/α1WT and KrasLA2/α1KO-Adα1 counterparts (Figs. 4A and 4B), suggesting a role for integrin α1β1 in anchorage-independent growth. Similarly, when cells were grown on collagen IV, thus primarily allowing the engagement of collagen binding integrins, only KrasLA2/α1WT and KRasLA2/α1KO-Adα1 tumor cells showed sustained proliferation (Fig. 4C).

Figure 4. Decreased anchorage-independent, anchorage-dependent and in vivo growth of KrasLA2/α1KO tumor epithelial cells.

Figure 4

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(A, B) The lung epithelial tumor cells indicated were plated onto soft agar as described in the Methods and colony number evaluated 7-8 days after plating. Values are means ± SD of one representative experiment performed in triplicate (5 fields/well were analyzed). (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/well) for further 48 hours and proliferation was then evaluated as described in the Methods. Values are means ± SD of one representative experiment performed in quadruplicate. (D) 2.5×105 or 1×106 KrasLA2/α1WT and KrasLA2/α1KO tumor cells were injected subcutaneously into nude mice (n=9) and tumor number and volume were analyzed 5 weeks post injection. Circles represent individual tumor volume, while the bar represents the mean.

To determine whether the differences in growth observed in vitro were reflected in vivo, the tumorigenic activity of KrasLA2/α1WT and KrasLA2/α1KO tumor cell populations was tested by injecting nude mice subcutaneously with various amounts of tumor cells. These assays were not performed with the KRasLA2/α1KO-Adα1 tumor cells as they only retain integrin α1 expression for ~ 7 days. As shown in Figure 4D, KrasLa2/α1WT 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/α1KO cells formed few small visible tumors only when injected in high numbers. Taken together these results confirm our finding that expression of integrin α1β1 plays a key role in the formation and growth of KrasLA2 lung tumors.

Reduced ERK activation accounts for decreased proliferation of KrasLA2/α1KO primary lung epithelial tumor cells

The ability of the tumor cells to activate ERK, p38 MAPK and Akt/PKB activation upon binding to collagen was determined as i) oncogenic Ras supports cell survival and proliferation by activating ERK (33) and down-regulating p38 MAPK activation (34, 35); ii) decreased ERK but increased p38 MAPK activation was observed in tumors derived from KrasLA2/α1KO mice (Figs. 2C and 2D); iii) activation of integrin α1β1 results in increased ERK phosphorylation via the Shc/Grb2 pathway (7); and iv) Kras promotes growth transformation by activation of the PI3K pathway (36). These experiments were performed on KrasLA2/α1WT and KrasLA2/α1KO tumor cells only as KrasLA2/α1WT and KrasLA2/α1KO-Adα1 behave in a similar fashion (see data above). As shown in Figure 5A, increased baseline activation of ERK but similar levels of phosphorylated Akt and p38 MAPK were evident in serum-starved KrasLA2/α1WT when compared to KrasLA2/α1KO tumor cells. Interestingly, when embedded within collagen gels, sustained ERK activation was only observed in KrasLA2/α1WT tumor cells, while it decreased significantly in KrasLA2/α1KO tumor cells within 15 minutes from plating. In contrast, p38 MAPK activation decreased over time in KrasLA2/α1WT tumor cells plated in collagen gels and it remained activated KrasLA2/α1KO tumor cells (Fig. 5A). Finally, comparable levels of collagen-mediated Akt activation were observed in both KrasLA2/α1WT and KrasLA2/α1KO tumor cells (Fig. 5A). Thus, sustained ERK activation and downregulation of p38 MAPK phosphorylation in the KrasLA2/α1WT tumor cells exposed to a collagenous environment requires the expression of integrin α1β1, suggesting that oncogenic Kras per se is not sufficient to regulate the activation state of these kinases.

Figure 5. Erk mediates KrasLA2/α1WT tumor epithelial cell growth on collagen substrata.

Figure 5

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(A) Equal number of serum-starved lung epithelial tumor cells was embedded in collagen I+IV gels for the time indicated (minutes). 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 Figure 4 in the presence or absence of the kinase inhibitors indicated. Values are the means ± SD of one representative experiment performed in quadruplicate. (*) represents statistically significant differences (p<0.05) between KrasLA2/α1WT and KrasLA2/α1KO cells; while (**) indicates statistically significant differences (p<0.05) between untreated and inhibitor-treated KrasLA2/α1WT cells.

To determine the contribution of ERK in KrasLA2/α1WT 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/α1WT 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/α1KO 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/α1WT or KrasLA2/α1KO tumor cells, strongly supporting a role for ERK, but not p38 MAPK in sustaining proliferation of KrasLA2/α1WT lung epithelial tumor cells.

DISCUSSION

The contribution of integrin α1β1 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 α1β1 expression by endothelial cells protects the host from tumor growth by reducing pathological angiogenesis. As it is unknown whether deleting the integrin α1 subunit also influences tumor initiation and formation, we crossed integrin α1-null mice with KrasLA2 mice, a model for spontaneous non-small cell lung cancer (25). We show that the KrasLA2/α1KO mice had a decreased incidence, number and size of primary lung adenomas resulting in a longer lifespan than KrasLA2/α1WT mice. Primary tumor cells derived from KrasLA2/α1KO mice had decreased tumorigenicity when injected into nude mice, formed fewer anchorage-independent colonies and proliferated less than KrasLA2/α1WT cells on collagen. These results demonstrate that integrin α1β1 plays a key role in both the development and growth of lung cancer, which is independent of integrin α1β1-mediated angiogenesis.

The KrasLA2 mouse model allowed us to demonstrate for the first time that the collagen binding receptor, integrin α1β1, 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 α1 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 α1β1 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/α1KO mice were also less vascularized than those derived from KrasLA2/α1WT mice, supports our previous finding that genetic ablation of integrin α1β1 results in decreased tumor-associated angiogenesis (9, 10, 12, 13) and integrin α1-blocking antibodies prevent growth-factor mediated angiogenesis in a corneal assay model (38).

Comparing primary lung epithelial tumor cells from both KrasLA2/α1WT and KrasLA2/α1KO mice allowed us to show that integrin α1β1 expression by the tumor cells played a critical role in the increased tumorigenicity seen in vivo. While 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 α1β1 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/α1KO 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 over-expression of the integrin α6 subunit (41). Thus, it is conceivable that integrin α1β1, together with oncogenic Kras, is responsible for regulating and supporting anchorage-independent growth of lung tumor epithelial cells.

Our in vitro signaling studies demonstrate that within collagenous environments sustained ERK but decreased p38 MAPK activation of KrasLA2 lung tumor cells requires the presence of integrin α1β1. 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 downregulated due to upregulation 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 α1β1 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 α1β1 is the only collagen receptor able to support cell survival and proliferation via activation of the Shc/Grb2/ERK pathway (7), while its loss results in increased p38 MAPK activation (28).

Our in vitro studies indicate that loss of integrin α1β1 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/α1WT and KrasLA2/α1KO mice. This result agrees with our previous finding that although primary α1-null endothelial cells fail to adhere and proliferate on collagenous substrata, no overall differences in vascular integrity are observed between wild type and integrin α1KO mice at baseline (9, 10).

In conclusion, we provide novel evidence that loss of the integrin α1 subunit decreases the incidence and growth of lung adenomas initiated by oncogenic Kras. As expression of integrin α1β1 has been detected in many types of human cancers; including a subset of patients with NSCLC (17), it is conceivable that Kras and integrin α1β1 cooperate to drive the growth of NSCLC in humans.

Supplementary Material

01

Supplementary Figure 1 Characterization of lung epithelial tumor cells derived from KrasLA2/α1WT and KrasLA2/α1KO mice. (A) Phase contrast image of lung epithelial tumors cells isolated from the mice indicated, showing epithelial-like morphology. Scale 10 μm. (B) Primary lung epithelial tumor cells were stained with anti-ZO1, anti-pan cytokeratins and anti-α smooth muscle actin antibodies. Mouse fibroblasts were used as control. Scale, 10 μm. (C) KrasLA2/α1KO tumor cells were infected with either the empty adenovirus (KrasLA2/α1KO-AdVo) or the human integrin α1 subunit cDNA (KrasLA2/α1KO-Adα1) as described in the Methods, and cell populations expressing the integrin α1 subunit were sorted by FACS.

ACKNOWLEDGMENTS

This work was supported by NCI/NIH R01 CA94849-01 (AP); RO1-DK 69921 and R01-DK075594 (RZ), a Merit award from the Department of Veterans Affairs (RZ); and NIH/Lung SPORE/P50CA090949 (D. Carbone).

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Associated Data

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Supplementary Materials

01

Supplementary Figure 1 Characterization of lung epithelial tumor cells derived from KrasLA2/α1WT and KrasLA2/α1KO mice. (A) Phase contrast image of lung epithelial tumors cells isolated from the mice indicated, showing epithelial-like morphology. Scale 10 μm. (B) Primary lung epithelial tumor cells were stained with anti-ZO1, anti-pan cytokeratins and anti-α smooth muscle actin antibodies. Mouse fibroblasts were used as control. Scale, 10 μm. (C) KrasLA2/α1KO tumor cells were infected with either the empty adenovirus (KrasLA2/α1KO-AdVo) or the human integrin α1 subunit cDNA (KrasLA2/α1KO-Adα1) as described in the Methods, and cell populations expressing the integrin α1 subunit were sorted by FACS.

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