Src-mediated morphology transition of lung cancer cells in three-dimensional organotypic culture
© Nguyen et al.; licensee BioMed Central Ltd. 2013
Received: 3 October 2012
Accepted: 11 February 2013
Published: 14 February 2013
A fribotic tumor microenvironment promotes progression of cancer. In this study, we utilize a reconstituted basement membrane mimics Matrigel based three-dimensional organotypic culture (rBM 3-D) to investigate the mechanisms that mediate the tumor promoting effects of the fibrogenic mediators TGF-β1 and type I collagen (Col-1) on lung adenocarcinoma cells. Similar to normal alveolar epithelial cells, the well-differentiated lung adenocarcinoma cells in rBM 3-D culture undergo acinar morphogeneis that features polarized epithelial cell spheres with a single central lumen. Either TGF-β1 or Col-1 modestly distorts acinar morphogenesis. On the other hand, TGF-β1 and Col-1 synergistically induce a transition from acinar morphology into stellate morphology that is characteristic of invasive and metastatic cancer cells. Inhibition of the Src kinase activity abrogates induction of stellate morphology, activation of Akt and mTOR, and the expression of tumor promoting genes by TGF-β1 and Col-1. To a similar extent, pharmacological inhibition of mTOR abrogates the cellular responses to TGF-β1 and Col-1. In summary, we demonstrate that TGF-β1 and Col-1 promote stellate morphogenesis of lung cancer cells. Our findings further suggest that the Src-Akt-mTOR axis mediates stellate morphogenesis. These findings also indicate that rBM 3-D culture can serve as an ideal platform for swift and cost-effective screening of therapeutic candidates at the interface of the tumor and its microenvironment.
KeywordsTGF-β1 Src Type I collagen Three-dimensional culture Extracellular matrix
A stiff and fibrotic microenvironment promotes tumor progression in experimental models [1, 2]. Accordingly, a fibrotic stroma is an independent prognostic indicator of metastasis and poor prognosis . The majority of such evidence comes from the investigation of breast cancer in which the aberrantly stiff extracellular matrix (ECM) is a well-established risk factor . A recent study has provided mechanistic insight into the link between the stiff ECM and progression of breast cancer . Lysyl-oxidase (LOX) increases the stiffness of ECM via crosslinking collagen and thereby enhances integrin signaling to promote invasion and metastasis . Recent advances in lung cancer research implicate a similar presence and function of a fibrotic tumor microenvironment. The expression of transforming growth factor-β1 (TGF-β1) and type I collagen (Col-1), two of the most potent fibrogenic mediators in the lung, is up-regulated in human lung cancer and overexpression of the two can promote invasion and metastasis in experimental models of lung cancer [6–8]. Elevated expression of LOX is a biomarker of invasion and an independent predictor of poor prognosis in patients with early stage lung adenocarcinoma . In experimental models of lung cancer, LOX promotes tumor progression and is targeted by the tumor suppressor gene LKB1 . However, the molecular mechanisms that mediate tumor progression promoted by the fibrotic tumor microenvironment in the lung remain poorly understood.
A substantial amount of our understanding of the tumor modulating functions of the tumor microenvironment has been obtained using three dimensional organotypic culture based on Matrigel, a reconstituted basement membrane mimics (rBM 3-D) [11–13]. rBM 3-D culture faithfully recapitulates salient in vivo properties of the epithelium from various tissues. The gene expression signature from rBM 3-D culture of breast cancer cells holds prognostic value for breast cancer . rBM 3-D culture is also a valuable tool to discriminate cancer cells with distinct tumorigenic potential . In general, the non-invasive/metastatic breast cancer cells exhibit a mixture of acinar and mass morphology that features spheroid colonies (mass) with occasional formation of a single central lumen (acinus), whereas the invasive/metastatic cancer cells exhibit stellate morphology that features prominent invasive projections that often bridge multiple cell colonies. More importantly, rBM 3-D culture provides an ideal system to reconstitute the tumor microenvironment for mechanistic investigations. For instance, investigation of Col-1 and its cognate integrin receptors in rBM 3-D culture of mammary epithelial cells has identified the stiff ECM-integrin axis as a driving force of initiation and progression of breast cancer [1, 2, 5]. Two recent applications of rBM 3-D culture demonstrate its promise in elucidating molecular and cell biology of lung epithelial cells. In rBM 3-D culture, primary human lung alveolar type II cells form alveolar acini . Similar to mammary epithelial cells, alveolar acini exhibit salient differentiation features, such as a polarized monolayer of alveolar type II cells and secretion of surfactant proteins into the central lumen. Because lung adenocarcinoma generally originates from alveolar type II cells, it is plausible that dysregulation of alveolar acini is a pivotal dedifferentiating step in lung tumorigenesis. In support of this concept, over-expression of the tumor suppressive PPAR-γ gene can restore alveolar acini in rBM 3-D organotypic culture of H2122 cells, an aggressive and poorly differentiated human lung adenocarcinoma cell line .
Recent advances have shown that the tumor associated stroma and microenvironment are active modulators of tumorigenesis rather than passive bystanders . The current study utilizes rBM 3-D organotypic culture to investigate a link between the behavior of lung cancer cells and the fribrogenic mediators derived from the tumor microenvironment.
Morphogenesis of lung cancer cells in rBM 3-D culture
Src-mediated stellate morphogenesis induced by TGF-β1 and Col-1
The Src kinase is a key signal transducer of ECM and growth factors . We then questioned whether the Src kinase activity is required for induction of stellate morphology by TGF-β1 and Col-1. To this end, A549 cells were exposed to TGF-β1 and Col-1 in the presence or absence of PP2 (5 μM), an Src selective inhibitor. When compared to the group treated with the DMSO vehicle, PP2 abrogated induction of stellate morphology by TGF-β1 and Col-1, but did not restore acinar morphology because the cell colonies were still void of a single central lumen (Figure 2C). Similar observations were made in A549LC cells upon exposure to various combinations of TGF-β1, Col-1, and PP2 (data not shown). To further confirm a requirement of the Src kinase activity for induction of stellate morphology by TGF-β1 and Col-1, we generated two variants of A549LC cells that were transduced with either a retroviral vector expressing a dominant-negative Src mutant (A549LCdnSrc) or its backbone vector (A549LCvec). Similar to PP2, the expression of the dnSrc mutant abolished stellate morphology induced by TGF-β1 and Col-1, whereas A549LCvec’s response to TGF-β1 and Col-1 was comparable to that of the parental A549LC cells (Figure 2, B & D). These findings indicated a requirement of the Src kinase activity for induction of stellate morphology by TGF-β1 and Col-1.
Activation of the Akt-mTOR axis
The current study investigates the molecular mechanisms that mediate cancer progression promoted by the fibrotic tumor microenvironment using rBM 3-D culture of lung cancer cells. We aim to define the molecular mechanisms that mediate the tumor-promoting effects derived from the fibrotic tumor microenvironment.
rBM 3-D culture has been successfully applied to characterize molecular and cell biology of normal and transformed mammary epithelial cells for the past two decades [11–13]. In essence, rBM 3-D culture bears similar potential for investigation of lung biology because the lung and the breast share several key developmental and structural traits, such as branching morphogenesis during development and formation of alveoli . Indeed, rBM 3-D culture of normal human lung alveolar type II epithelial cells promotes expression of the differentiation markers, such as surfactant protein C and formation of acini, an in vitro mimic of alveoli . More importantly, over-expression of PPAR-γ, a tumor suppressor gene, can restore formation of acini in a poorly differentiated human lung cancer cell line in rBM 3-D culture . Our findings strengthen the concept that rBM 3-D culture can be used to assess invasive and metastatic potential of lung cancer cells by comparing morphogenesis of four lung cancer cell lines with distinct tumorigenic behaviors in vivo. By and large, the well-differentiated lung adenocaricnoma cells, A549 and mK-ras-LE, form acini, whereas the more aggressive A549LC and LLC cells exhibit mass and stellate morphology (Figure 1). The diverse growth patterns of these four lung cancer cell lines in rBM 3-D culture are congruent to their disparate histology and tumorigenic potential in vivo (Figure 1). It is noteworthy that rBM 3-D culture reveals distinct morphogenesis between A549 (acinar morphology) and A549LC (mass morphology), whereas the two cell lines appear nearly identical in 2-D culture (data not shown). The morphological difference in rBM 3-D is also congruent to their distinct histology and tumorigenic activity in vivo (Figure 1, A & B). With further optimization and validation, rBM 3-D organotypic culture can be utilized as a surrogate to qualitatively and quantitatively assess tumorigenic properties of lung cancer cells.
One major advantage of rBM 3-D culture is that it allows assessment of tumor modulating cues derived from the tumor microenvironment [11–13]. As revealed in our study, TGF-β1 and Col-1 synergistically induce stellate morphology, a hallmark feature of invasive/metastatic cancer cells (Figures 1 & 2) . This combined exposure may recapitulate the fibrotic tumor microenvironment in vivo where lung cancer cells are simultaneously and constantly exposed to a variety of fibrogenic mediators [6–8]. Induction of stellate morphology by a combination of TGF-β1 and Col-1 is also consistent with a previous study in which provisional ECM, such as fibronectin and Col-1 potentiates epithelial-mesenchymal transition (EMT) of alveolar type II epithelial cells in response to TGF-β1 in 2-D culture . Thus, stellate morphology induced by TGF-β1 and Col-1 can be perceived as a phenomenon of EMT in rBM 3-D culture, which allows investigation of EMT of lung cancer cells, a pivotal step towards invasion/metastasis in the context of ECM. In support of our notion, characterization of EMT using rBM 3-D culture has been proposed as a routine protocol based on initial success of this approach .
Our attempt to pinpoint the mediators of the synergistic induction of stellate morphology by TGF-β1 and Col-1 results in limited success. Nevertheless, we identify the signaling pathway and target genes activated by the TGF-β1 arm, which is not sufficient, but required for transition from acinar to stellate morphology (Figures 2, 3, 4). Specifically, the Src kinase activity is required for induction of stellate morphology and activation of gene expression by TGF-β1 in the presence or absence of Col-1 (Figures 2 & 3). Similarly, the Src kinase activity appears to be essential for activation of the Akt-mTOR axis by TGF-β1 in the presence or absence of Col-1 (Figures 2 and 4). Besides the inducible stellate morphogenesis, the Src kinase activity appears to be required for native stellate morphogenesis of the invasive/metastatic cancer cell lines because inhibition of the Src kinase activity abrogates stellate morphogenesis of the invasive/metastatic LLC, 4T1, and MDA-MB231 cells (unpublished observations).
Despite similar distortion of acinar morphogenesis, only TGF-β1, but not Col-1 stimulates the expression of the MYC, LOX, and PAI-1 (Figures 1 & 3). It is conceivable that Col-1 employs an alternative gene expression program to disrupt acinar morphogenesis. In support of this notion, Col-1 stimulates the expression of the oncogenic miR-21 gene in rBM 3-D culture, which is not observed in lung cancer cells exposed to TGF-β1 (unpublished observations) . Among the TGF-β1-activated tumor promoting genes, LOX exhibit an Src- and mTOR-dependence and a strong correlation to stellate morphology (Figures 3 & 4) . These findings suggest a novel mechanism for the elevated expression of LOX in human lung cancer in that TGF-β1 induces the expression of LOX in lung cancer cells via the Src-Akt-mTOR axis. It is also conceivable that the TGF-β1-induced expression of LOX in rBM 3-D culture crosslinks the supplemented Col-1 to substantially increase the stiffness of rBM 3-D culture and thereby mediates synergistic induction of stellate morphology by TGF-β1 and Col-1. Among the three genes examined upon blockade of Src and mTOR, PAI-1 appears to be refractory to inhibition of mTOR, whereas inhibition of Src diminishes activation of all three genes (Figures 3 & 4). This suggests that mTOR mediate only part of the gene activation program activated by Src upon exposure to TGF-β1. This observation could also be attributed to the SMAD3 binding motif in the PAI-1 promoter that induces the expression of PAI-1 through SMADs, the canonical TGF-β pathway and delivers resistance to blockade of the non-canonical TGF-β pathways, such as mTOR .
In summary, we demonstrate that the fibrogenic mediators derived from the tumor microenvironment promote stellate morphogenesis of lung cancer cells. Our results further suggest that the Src-Akt-mTOR axis, a group of promising therapeutic targets in lung cancer, acts as a signal transducer of the fibrotic tumor microenvironment [22, 23, 31]. Our work warrants further investigation to elucidate the molecular mechanisms that mediate synergistic induction of stellate morphology by TGF-β1 and Col-1. These findings also strongly suggest that rBM 3-D culture can serve as an ideal platform for swift and cost-effective screening of therapeutic candidates at the interface of the tumor and its microenvironment.
Reagents and plasmids
PP2, an Src specific inhibitor, was purchased from Calbiochem (San Diego, CA). Matrigel was purchased from BD Biosciences (Rockville, MD). Rat Col-1 was purchased from Sigma (St. Louis, MO). Recombinant human TGF-β1 was obtained from R&D Systems (Minneapolis, MN). A dominant-negative chicken Src-K295R mutant expressing retroviral vector (dnSrc) and its backbone (pLNCX) were kindly provided by Dr. Brugge at Harvard University [32, 33]. Torin1, an mTOR-specific inhibitor was a gift from Dr. Sabatini at MIT . Invitrogen (Carlsbad CA) provided the antibodies specific for total and phosphorylated (Tyr861) FAK. Cell Signaling (Danvers MA) provided the antibodies specific for total and phosphorylated Src (Tyr416), Akt (Ser473), mTOR (Ser2448), and p70 S6K (Thr389).
A549 cells, a human lung adenocarcinoma cell line were obtained from ATCC (Manassas VA) and cultured as previously described [34, 35]. A549LC cells were derived from parental A549 cells using a murine model of lung metastasis [36, 37]. Briefly, A549 cells (106 cells/mouse) were injected via the jugular vein into adult female beige-SCID mice (Charles River). Four months after injection, lungs were inspected and one metastatic nodule was excised, disaggregated and established in culture. The dnSrc expressing variant of A549LC (A549LCdnSrc) and its matching backbone vector variant (A549LCvec) were generated using retroviral transduction as we previously described . mK-ras-LE cells, a murine lung epithelial cell line, were established from a tumor bearing lung of a K-ras LA1 transgenic mouse and cultured in RPMI-1640 as described elsewhere [20, 29]. Lewis lung carcinoma cells (LLC), a metastatic murine lung cancer cell line, were purchased from ATCC (Manassas, VA) and cultured in DMEM.
rBM 3-D organotypic culture and image analysis
rBM 3-D organotypic culture was employed because of the prior success of this approach in characterizing differentiation of both primary and transformed lung epithelial cells [11–13, 16, 17]. Briefly, the lung cancer cells were seeded in an overlay fashion on a layer of Matrigel on day zero. The culture medium containing 4% Matrigel (volume/volume) was replaced every other day. Formation of acini was monitored for twelve days prior to harvest for image, RNA, and protein analyses. The cultured cells were visualized using fluorescent staining for filamentous actin with Alexa 488 conjugated phalloidin (Invitrogen, Carlsbad CA). The images were captured using confocal fluorescent or phase contrast microscopy as we previously described [26, 39]. In the selected cultures, various combinations of TGF-β1 (5 ng/ml), Col-1 (1.5 μg/ml), and Torin-1 (250 nM) were added to rBM 3-D culture.
RNA extraction and quantitative RT-PCR
Total cell RNA was extracted from rBM 3-D culture using TRIzol per the provider’s instructions (Invitrogen, Carlbad CA). The expression of each gene of interest was determined using quantitative RT-PCR on an iCycler (BIO-RAD, Hercules CA) and compared across the groups as described else where . The sequences of each pair of primers were listed in Additional file 1: Table S1.
Total cell protein was extracted using RIPA buffer supplemented with Protease and Protein Phosphatase Inhibitor Cocktails (Sigma, St. Louis MO) after A549 cells and their variants were extracted from rBM 3-D culture . The expression of the total and phosphorylated proteins of interest was determined using immunoblots as described we previously described .
Implantation of lung cancer cells
All mouse studies were carried out following the animal protocol approved by the Institute Animal Care and Use Committee at Tulane University School of Medicine. Subcutaneous implantation of human and mouse lung cancer cells (2 × 106 cells/mouse) into male nude and syngeneic mice (Charles River, Wilmington, MA) was carried out as we previously described [36, 41]. Each group of tumor graft consisted of 7 mice. Tumor growth was monitored daily after implantation. The tumor mass was dissected from mice at four weeks after implantation and processed for weighing and H&E staining.
When presented, means and standard deviations were obtained from 3 independent experiments. A P value between any two selected groups was determined using unpaired two-tailed Student's T-test (GraphPad Prism, Version 5).
Transforming growth factor-β1
Type I collagen
Mammalian target of rapamycin
Plasminogen activator inhibitor-1.
This work is supported by Tulane Research Enhancement Fund. We are grateful to Dr. Brugge and Dr. Sabatini for providing the Src mutant and the mTOR inibitor, respectively.
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