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[Cancer Research 61, 2439-2444, March 15, 2001]
© 2001 American Association for Cancer Research

Biochemistry and Biophysics

Tyrosine Kinase-dependent, Phosphatidylinositol 3'-Kinase, and Mitogen-activated Protein Kinase-independent Signaling Pathways Prevent Lung Adenocarcinoma Cells from Anoikis1

Lin Wei, Yu Yang and Qiang Yu2

Pulmonary Center, Department of Medicine, and Department of Biochemistry, Boston University Medical Center, Boston, Massachusetts 02118


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Normal epithelial cells are anchorage-dependent. Detachment of normal epithelial cells from their substratum causes apoptosis, termed anoikis. Malignant tumor cells, however, can survive and proliferate independent of anchorage. To understand the molecular basis of tumor cell anchorage independence, we investigated the role of tyrosine kinases and their downstream signaling pathways in anoikis resistance of human lung adenocarcinoma cells. Four of the five lung adenocarcinoma cell lines analyzed are resistant to anoikis. Tyrosine kinase inhibitor genistein rendered three of them sensitive to anoikis. Although cell detachment induced rapid protein tyrosine dephosphorylation in Madin-Darby canine kidney cells, a nontransformed epithelial cell line, tyrosine phosphorylation of several proteins in the tumor cells is anchorage-independent. Similarly, phosphorylation of Akt and mitogen-activated protein kinase, two signaling proteins downstream of tyrosine kinases, was decreased in Madin-Darby canine kidney cells but increased in some of the tumor cells upon cell detachment. Inhibition of phosphorylation of the two proteins, however, did not induce anoikis in the tumor cells. Specific inhibitors to several known tyrosine kinases also failed to induce anoikis in these cells. These data suggest the existence of tyrosine kinase-dependent phosphatidylinositol 3'-kinase and mitogen-activated protein kinase-independent signaling pathways that function to regulate cell survival and death. Alteration in these pathways may count for the anchorage-independent survival of the lung adenocarcinoma cells and other malignant tumor cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Survival, growth, and differentiation of normal epithelial cells are anchorage-dependent. Detachment of normal epithelial cells from their substratum causes apoptosis, termed anoikis (1 , 2) . This mechanism is crucial for maintaining homeostasis, organization, and architecture of epithelia of different organs (3, 4, 5) . The cellular signals generated by the cell-substratum interactions are transduced into cells through their cell surface receptors. The best-characterized cell survival and growth-signaling pathway that transmits the cell-matrix interaction-generated signals is the integrin-signaling pathway (2 , 6 , 7) . The key mechanism for the integrin-mediated signal transduction is the activation of intracellular tyrosine kinases such as FAK3 (2 , 8 , 9) . Engagement of integrins induces phosphorylation and activation of tyrosine kinases. Activated tyrosine kinases recruit and phosphorylate intracellular signaling adaptor proteins and trigger a number of signaling pathways that regulate cell survival and growth. The importance of the integrin/tyrosine kinase signaling pathway in cell anchorage-dependent survival and growth has been demonstrated by introducing constitutively active form of FAK into the anchorage-dependent cells and render them anchorage-independent (10) . Two major cell survival and growth signaling pathways downstream of tyrosine kinases are the PI 3K-Akt pathway and the MAPK pathway (2 , 9 , 11, 12, 13) . The activities of both PI 3K and MAPK are regulated by integrin-regulated tyrosine kinases. Transfection of cells with constitutively active forms of PI 3K or Akt blocks anoikis, whereas inhibition of PI 3K induces anoikis (14 , 15) . The MAPK pathway has also been shown to be involved in regulating cell survival (13 , 16 , 17) . Therefore, tyrosine kinases, PI 3K-Akt, and MAPK-activated signaling pathways are crucial for mediating cell survival signals from cell-matrix interactions.

The survival and growth of malignant tumor cells, on the other hand, are anchorage-independent. They can survive and grow without attaching to their substratum (18 , 19) . Although this property of tumor cells has been known for decades and has been considered as one of the hallmarks of malignant tumor cells, the molecular mechanism underlying this property of tumor cells has not been completely understood. Our understanding of the molecular mechanism of tumor cell anchorage independence comes mainly from studies of transforming activities of tumor viruses and oncogenes. Many tumor viruses and oncogenes confer anchorage-dependent cells with anchorage independence when they are introduced into these cells (1 , 14 , 18 , 20) . One important activity of various oncogenes is that they activate the signaling pathways downstream of integrin and integrin-activated tyrosine kinases; e.g., it has been shown in MDCK cells that oncogenic ras confers cells with anchorage independence by activating the PI 3K-Akt pathway (1 , 14) . In doing so, the oncogene-transformed cells circumvent the requirement of integrin-mediated attachment in generating the necessary cell survival and growth signals. Because the PI 3K-Akt and MAPK pathways can be activated by many different tyrosine kinases, any genetic mutations that can activate these tyrosine kinases may confer anchorage independence to the cells as well. However, the significance of tyrosine kinases and their downstream PI 3K and MAPK signaling pathways in preventing human cancer cells from anoikis has not been thoroughly investigated. It will be very interesting to explore additional pathways that are critical for tumor cells to prevent anoikis.

We have analyzed a panel of lung adenocarcinoma cell lines for their resistance to anoikis and explored the role of tyrosine kinases, PI 3K-Akt, and MAPK in their resistance to anoikis. We found that the anoikis resistances of most of the lung tumor cells we analyzed were tyrosine kinase-dependent. PI 3K, Akt, and MAPK did not play significant roles in mediating the cell survival signals from the tyrosine kinases that protect the tumor cells from anoikis, although the regulation of Akt and MAPK were altered in some of the tumor cells. Therefore, tyrosine kinase-dependent signaling pathways other than PI 3K-Akt and MAPK pathways may exist and are responsible for the tumor cells anchorage-independent survival.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Anoikis Assay.
MDCK cells, MCF-10A cells, and the five human lung adenocarcinoma cell lines were purchased from the American Type Culture Collection. MCF10A cells were maintained in MEGM medium (Clonetics). All of the other cell lines were maintained in DMEM (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum.

The anoikis assay was essentially performed as described by Frisch and Francis (1) . Briefly, cells were grown to confluence in 100-mm tissue culture dishes. Trypsinized cells were then counted, and 106 cells were plated onto 60-mm polyHEMA-coated Petri dishes. The polyHEMA-coated dishes were prepared by applying 2-ml polyHEMA solution [10 mg/ml polyhydroxyethylmethacrylate (Aldrich Chemical Co., Milwaukee, WI) in ethanol] onto the dish, drying in the tissue culture hood, and repeating once, followed by extensive wash with PBS (>3 times). After culturing for various time periods, cells were collected from the polyHEMA dishes by pipetting or from tissue culture dishes by scraping. Cytosolic nucleic acids, which contain both fragmented genomic DNA and RNA, were extracted with a 0.6-ml solution of 0.5% Triton X-100, 10 mM EDTA, and 10 mM Tris (pH 7.4), pheno-chloroform extracted three times, and ethanol precipitated and analyzed on a 1.5% agarose gel. The gel was incubated in RNase A-containing solution (5 µg/ml) to digest away the RNAs in the gel before photographing.

Treatment of Cells with Various Inhibitors.
Tyrosine kinase inhibitor genistein and tyrosine phosphatase inhibitor sodium orthovanadate were purchased from Sigma Chemical Co. (St. Louis, MO). PI 3K inhibitor wortmannin and LY294002, MEK inhibitor PD98059, and specific tyrosine kinase inhibitor tyrphostins were purchased from Calbiochem (La Jolla, CA). Stock solution of sodium orthovanadate was constituted in H2O at a concentration of 50 mM adjusted to pH 10, boiled until it became translucent and the pH was readjusted to pH 10. The rest of the various inhibitors were dissolved in DMSO. In experiments that cells were treated with the various inhibitors, the same volumes of corresponding solvents were added to the controls.

Antibodies and Immunoblot Analysis.
A mouse monoclonal antibody to phosphotyrosine (4G10) was obtained from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal antibodies to phospho-Akt, Akt, phospho-MAPK, and MAPK were from New England BioLabs (Beverly, MA). Antibodies purchased commercially were used as recommended by the manufactures.

For immunoblot analysis, cells were lysed in a modified RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.5% (w/v) sodium deoxycholate, 0.1% SDS, 0.2 mM phenylmethylsulfonyl fluoride, leupeptin (5 µg/ml), aprotinin (5 µg/ml), and 1 mM Na3VO4]. Cell nuclei were removed from lysates by centrifugation for 10 min. Protein concentration was determined with the Bradford reagent (Bio-Rad). Proteins were resolved by SDS-PAGE (7.5%) and transferred to a nitrocellulose membrane. For immunoblot analysis with monoclonal antibody 4G10 to phosphotyrosine, nonspecific sites on the membrane were blocked by incubation for 20 min at room temperature with 3% (w/v) nonfat dry milk in PBS. For immunoblot analysis with other antibodies, the membrane was incubated for 1 h at room temperature in a solution containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% (v/v) Tween 20. All of the membranes were then incubated overnight at 4°C with primary antibodies in the same blocking solution. The membranes were then washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (Promega). Immune complexes were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lung Adenocarcinoma Cells Are Generally Resistant to Anoikis.
We analyzed five human lung adenocarcinoma cell lines, A549, SK-LU-1, NCI-H23, NCI-H1792, and Calu-3, all of which normally grow as attached monolayer cells, for their sensitivity to anoikis. Two immortalized but nontransformed and anoikis-sensitive epithelial cell lines were used as controls. One is the MDCK cell line (1) , and the other is the human mammary gland cell line MCF-10A (21) . All of the cell lines were cultured into confluence in regular tissue culture dishes before anoikis analysis. After the cells became confluent, they were trypsinized, transferred into polyHEMA-coated dishes, which prevent cells from attachment (1) , and cultured for 15 to 48 h. Cells were then harvested, and the cytosolic low molecular weight DNA from these cells were isolated and analyzed on agarose gel. Anoikis was evaluated by the appearance of fragmented cytosolic DNA on the agarose gel. The control MDCK cells underwent marked apoptosis when cultured on polyHEMA-coated dishes, as evidenced by the appearance of fragmented DNA on the agarose gel (Fig. 1)Citation . Cell death became evident after 7-h culturing on polyHEMA dishes (data not shown). The MCF-10A cells also underwent apoptosis upon culturing in suspension, although the DNA laddering was not as obvious as that of the MDCK cells (Fig. 1)Citation . Four of the five tumor cell lines tested, A549, SK-LU-1, NCI-H23, and NCI-H1792, were resistant to anoikis. No apparent DNA fragmentation occurred in these cells after 15 to 48 h culturing on polyHEMA-coated dishes (Fig. 1Citation and data not shown). Low levels of high molecular weight cytosolic DNA appeared in some of the tumor cells (Fig. 1)Citation . However, the amount of cytosolic DNA from these cells was much less than that of the control cells, and there was no obvious laddering appearance of the fragmented DNA in these cells. One of the tumor cell lines, Calu-3, showed low levels of DNA fragmentation, suggesting that this cell line is partially resistant to anoikis (Fig. 1)Citation .



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  		Fig. 1. Anoikis analysis of human lung adenocarcinoma cells. MDCK, MCF-10A, and five human lung adenocarcinoma cell lines were cultured on regular tissue culture dishes until confluence. The cells were then trypsinized and counted. 106 cells were seeded in polyHEMA-coated Petri dishes and cultured for 15 or 48 h, as indicated. Controls are confluent cells harvested directly from the regular tissue culture dishes (0 h). Cytoplasmic DNAs were isolated from these cells and analyzed by agarose gel electrophoresis as described in "Methods and Materials." DNA from an equal amount of cells was loaded in each lane. M, the 1-kb DNA marker from Life Technologies, Inc.

 
These data indicate that the tumor cells are generally resistant to anoikis in comparison to nontransformed epithelial cells. The extent of the resistance varies between different tumor cells, suggesting that there may be qualitative and/or quantitative differences in the altered molecules and signaling pathways between different tumor cells. Cells that are highly resistant to anoikis may harbor mutations in key molecules of cell survival/death pathways.

Function of Protein Tyrosine Phosphorylation and Tyrosine Kinases in Tumor Cell Anoikis Resistance.
Because many cell survival signals are generated through tyrosine kinases, which include integrin-regulated tyrosine kinases and growth factor receptor tyrosine kinases, we investigated the importance of tyrosine kinases and protein tyrosine phosphorylation in tumor cell resistance to anoikis by inhibiting the tyrosine kinases using tyrosine kinase-specific inhibitors. The addition of tyrosine kinase inhibitor genistein induced anoikis in three of the four anoikis-resistant tumor cells. The extent of cell death induced by genistein varies between the different cell lines, with A549 as the most sensitive cell line. One of the tumor cell lines, SK-LU-1, was insensitive to genistein treatment (Fig. 2A)Citation .



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  		Fig. 2. Induction of anoikis by tyrosine kinase inhibitor and reversal by tyrosine phosphatase inhibitor. Confluent cells were trypsinized and seeded in the polyHEMA-coated dishes and cultured for 15 h in the presence or absence of tyrosine kinase inhibitor genistein (A) and genistein plus tyrosine phosphatase inhibitor sodium vanadate (B), as indicated. Cytoplasmic DNAs were then isolated from these cells and analyzed by agarose gel electrophoresis. M, the 1-kb DNA marker from Life Technologies, Inc.

 
To confirm that the cell death induced by genistein was indeed because of inactivation of tyrosine kinases, we added tyrosine phosphatase inhibitor sodium vanadate to the cells together with genistein. Addition of vanadate alleviated the genistein-induced apoptosis in a dose-dependent manner (Fig. 2BCitation and data not shown), indicating that a constant level of tyrosine phosphorylation of certain proteins is essential for the survival of the tumor cells when cultured in suspension. These data clearly demonstrated that activated tyrosine kinases are important for the tumor cell resistance to anoikis. Because the degree of DNA fragmentation induced by genistein in the tumor cells is still significantly less than that seen in the suspended MDCK cells, the data also suggested that activated tyrosine kinases may play only a partial role in preventing these tumor cells from anoikis.

To identify which tyrosine-phosphorylated proteins in the tumor cells might be responsible for their resistance to anoikis, we examined the general pattern of protein tyrosine-phosphorylation in the three genistein-sensitive tumor cells and compared them with that of MDCK cells under different culturing conditions. There were three major tyrosine-phosphorylated protein bands present in the control anoikis-sensitive MDCK cells (Fig. 3)Citation . All of the three protein bands underwent dephosphorylation when the cells were cultured in suspension, indicating that most of the protein tyrosine phosphorylation in the MDCK cells is anchorage-dependent. The protein tyrosine phosphorylation patterns in the three tumor cells were quite different from that of the MDCK cells. First, there were generally more tyrosine-phosphorylated proteins present in the tumor cells, particularly in A549 cells. Second, although some of the proteins in the tumor cells underwent dephosphorylation upon cell detachment, there were still considerable numbers of tyrosine-phosphorylated proteins present in the cells after 24 h culturing in suspension (Fig. 3)Citation . Third, the amount of tyrosine-phosphorylation correlates with the sensitivity of the cells to genistein treatment (Fig. 2ACitation and Fig. 3ACitation ). Furthermore, genistein treatment inhibited the phosphorylation of a majority of the tyrosine-phosphorylated proteins in the tumor cells. Taken together, these data strongly suggest the existence of tyrosine kinase-mediated cell survival signals in the tumor cells that are responsible for the anchorage-independent survival of the tumor cells.



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  		Fig. 3. Changes in protein tyrosine phosphorylation upon cell detachment. Confluent cells were trypsinized and cultured as attached cells in regular cell culture dishes (att) or in suspension in polyHEMA-coated dishes with (sus+G) or without (sus) genistein for 24 h. The cells were then harvested and lysed in RIPA protein lysis buffer. Equal amounts of proteins were loaded in each lane and resolved by SDS-PAGE, followed by immunoblot analysis using antiphosphotyrosine antibody 4G10 ({alpha}P-Tyr). The protein molecular weight markers are in kDa.

 
Function of Specific Tyrosine Kinases, Akt, and MAPK in Tumor Cell Anoikis Resistance.
To identify the specific tyrosine kinases and to define the signaling pathways that are responsible for anoikis resistance of the different tumor cells, we focused on one of the tumor cell lines, the A549 cell line, for further analysis, because the A549 cells contain the highest level of tyrosine-phosphorylated proteins when cultured in suspension and the A549 cell line is one of the most sensitive cell lines for genistein-induced anoikis (data not shown).

We tried a panel of specific tyrosine kinase inhibitor tyrphostins for their ability to induce anoikis. These include the inhibitors to epidermal growth factor receptor [AG213 (22) , AG1478 (15) , AG18 (23) , and AG527 (24) ], platelet-derived growth factor receptor [AG1296 (25) and AG17 (26) ], Src [AG1478 (27) and AG10 (28) ], and Abl [AG957 (29) ]. The specific inhibitors to these kinases were added to the A549 cells cultured in suspension individually, at concentrations that were at least 10 times higher than their corresponding IC50, as well as in a mixture. Although the addition of the inhibitors inhibited cell proliferation, as evidenced by the reduced cell number after 48-h culturing, none of them induced anoikis of the tumor cells at 15 h (Fig. 4)Citation or at 48 h (data not shown). These data suggest that the tyrosine kinases inhibited by these inhibitors are not the major players in preventing A549 cells from anoikis.



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  		Fig. 4. Effect of specific tyrosine kinase inhibitors on A549 cells. A, effects of the inhibitors on cell growth. A549 cells were cultured in suspension in the absence (C) or presence of specific tyrosine kinases inhibitors AG10 (450 µM), AG18 (400 µM), AG213 (50 µM), AG490 (50 µM), AG527 (50 µM), AG17 (50 µM), AG957 (50 µM), AG1296 (50 µM), or AG1478 (50 µM), individually, or as a mixture of 50 µM each (Mix) for 48 h. Cells were then trypsinized, and the number of the cells from each dish were counted and plotted. B, effects of the inhibitors on cell apoptosis. An equal number of A549 cells were cultured in suspension in the absence (C) or presence of the various inhibitors for 15 h. Cells were harvested by centrifugation, and the cytosolic DNAs were isolated and analyzed by agarose gel electrophoresis. M, the 1-kb DNA marker from Life Technologies, Inc.

 
We next investigated the involvement of known signaling molecules downstream of tyrosine kinases in mediating the cell survival signals from the unidentified tyrosine kinases in A549 and other tumor cells. PI 3K-Akt pathway is a key signaling pathway in transmitting many tyrosine kinase-generated, including matrix-induced, cell survival signals (14) . To investigate whether PI 3K and Akt also play a role in anoikis resistance of the A549 tumor cells, we analyzed the regulation and function of PI 3K and Akt in MDCK cells during anoikis and compared them with that of the A549 tumor cells. The activity of the PI 3K/Akt pathway is monitored by the phosphorylation of Akt. Akt in both MDCK and A549 cells was phosphorylated when the cells were cultured in regular tissue culture dishes. Upon cell detachment, the Akt in MDCK cells underwent rapid dephosphorylation (Fig. 5A)Citation . In contrast, the phosphorylation of Akt in A549 cells was increased upon culturing in suspension (Fig. 5A)Citation , suggesting that Akt and its upstream PI 3K are not inactivated but rather activated upon cell detachment in the tumor cells.



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  		Fig. 5. Regulation of Akt and MAPK during cell detachment and their function in tumor cell anoikis resistance. A, regulation of phosphorylation of Akt and MAPK during cell detachment and their inhibition by wortmannin and PD98059. Confluent MDCK and A549 cells were either lysed directly in RIPA buffer (att) or trypsinized and cultured in polyHEMA-coated dishes in the presence or absence of wortmannin (wort; 1 µM) or PD98059 (50 µM) for 6 h and then harvested and lysed in RIPA protein lysis buffer (sus). An equal amount of proteins were loaded in each lane and resolved by SDS-PAGE, followed by immunoblot analysis using anti-phospho-Akt ({alpha}Akt-P), anti-Akt ({alpha}-Akt), anti-phospho-MAPK ({alpha}-MAPK-P), and anti-MAPK ({alpha}-MAPK) antibodies sequentially. B, effect of wortmannin, LY294002, and PD98059 on apoptosis of A549 cells. Confluent A549 cells were trypsinized and cultured in polyHEMA-coated dishes in the presence or absence of wortmannin (0.5 µM), LY294002 (20 µM), or PD98059 (50 µM) for 15 h. Wortmannin was added twice at 8-h intervals. Cells were harvested by centrifugation, and the cytosolic DNAs were isolated and analyzed by agarose gel electrophoresis. C, effect of wortmannin, LY294002, and PD98059 on phosphorylation of Akt and MAPK. One third of the above treated A549 cells were lysed in RIPA protein lysis buffer. An equal amount of proteins were loaded in each lane and resolved by SDS-PAGE, followed by immunoblot analysis using anti-phospho-Akt ({alpha}Akt-P), anti-Akt ({alpha}-Akt), anti-phospho-MAPK ({alpha}-MAPK-P), and anti-MAPK ({alpha}-MAPK) antibodies sequentially.

 
We then investigated the regulation of MAPK, the other major downstream player in tyrosine kinase-mediated signaling, during cell detachment. The regulation of MAPK upon cell detachment was very similar to that of Akt. It was dephosphorylated in MDCK cells, whereas it was hyperphosphorylated in A549 cells when they were cultured in suspension (Fig. 5A)Citation .

These data indicate that both Akt and MAPK in A549 cells are activated upon cell detachment, suggesting an activation of kinases or an inactivation of phosphatases upstream of the two enzymes in the A549 tumor cells when they are detached from their substratum.

To test whether the increased activation of PI 3K/Akt and MAPK are responsible for the tumor cell anoikis resistance, we inactivated PI 3K and MAPK in the tumor cells with PI 3K-specific inhibitors wortmannin and LY294002 and MEK inhibitor PD98059, respectively. Addition of wortmannin, LY294002, and PD98059 abolished the phosphorylation of Akt and MAPK completely (Fig. 5A)Citation . However, they did not induce apoptosis of the tumor cells (Fig. 5B)Citation . We also tested the effect of wortmannin, LY294002, and PD98059 on the other four tumor cells. None of them were induced to undergo significant apoptosis by the inhibitors (data not shown). These data suggest that although the regulation of PI 3K and MAPK were altered in some of the tumor cells, presumably caused by alterations in the upstream tyrosine kinases or phosphatases, they are not essential for preventing the tumor cells from anoikis.

Taken together, these data suggest that PI 3K and MAPK-independent but tyrosine kinase-dependent signaling pathways are present in the lung adenocarcinoma cells that are responsible for preventing the tumor cells from anoikis.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tumor cell anchorage independence is a hallmark of tumor cells and has been known for many years. The molecular mechanism underlying this phenomenon has mainly been studied by introducing known oncogenes into immortalized cell lines to examine their ability to confer anchorage independence to those cells. In this way, oncogenes, e.g., Ras, Src, and their downstream-signaling molecules, such as PI 3K-Akt and MAPK, have been identified as critical players in compensating the cell survival and growth signals normally provided by the cell-matrix interaction (1 , 14) . However, it is not clear whether these pathways are the sole players in transmitting cell survival signals to prevent the naturally occurring cancer cells from anoikis. The molecules and signaling pathways in anoikis resistance of human cancer cells have not been adequately explored. We investigated the mechanism of tumor cell anoikis resistance by studying the regulation and function of tyrosine kinases and several key cell survival signal transducers in anoikis-resistant human lung adenocarcinoma cells and compared them with that of anoikis-sensitive noncancerous cells. We found that certain anchorage-independent tyrosine kinase activities in the tumor cells are critical for tumor cells to survive after detachment. Tyrosine kinase-specific inhibitor genistein induced cell death in most of the lung adenocarcinoma cell lines tested. Furthermore, the induction of cell death by genistein is alleviated by the addition of tyrosine phosphatase inhibitors simultaneously, confirming that it is indeed the tyrosine kinase activities that prevent the tumor cells from anoikis. It also suggests that a minimal level of tyrosine phosphorylation of the unidentified proteins is essential for maintaining the cell survival signals. In this regard, it is noteworthy that although tyrosine phosphorylation of most of the proteins in the anoikis-sensitive MDCK cells is anchorage-dependent, tyrosine phosphorylation of several proteins in the tumor cells, particularly in the A549 cells, is anchorage-independent. Identification of these proteins and the tyrosine kinases responsible for the phosphorylation of these proteins may reveal new tyrosine kinase-mediated cell survival signaling pathways.

One of the known signaling pathways downstream of various tyrosine kinases that transmit cell survival signals is the PI 3K-Akt pathway, which has been shown as the major pathway to mediate cell survival signals from integrin-FAK as well as many peptide growth factor receptors (2 , 11 , 12) . Therefore, we investigated the possibility that the tyrosine kinase-dependent cell survival signals in the lung adenocarcinoma cells are also mediated through the PI 3K-Akt pathway. We found that although the phosphorylation and, hence, the activity of Akt remain to be high or even increased instead of decreased in some of the tumor cells when they are cultured in suspension, inhibition of Akt phosphorylation by PI 3K specific inhibitors did not induce cell death, suggesting that the PI 3K-Akt pathway is not the major pathway or at least not the only pathway in mediating the cell survival signals from the tyrosine kinases in the tumor cells. Another major signaling pathway downstream of the tyrosine kinases is the MAPK pathway. Although the activity of MAPK is regulated by cell adhesion, its role in cell survival, particularly in integrin-FAK-mediated cell survival, is less prominent than that of the PI 3K-Akt pathway (15) . Similar to Akt, the phosphorylation of MAPK is also increased in some of the lung adenocarcinoma cells upon cell detachment, suggesting that both PI 3K and MAPK are similarly regulated in the tumor cells. Inhibition of MAPK activation by the MEK inhibitor did not induce a significant level of anoikis, suggesting that MAPK does not mediate the cell survival signals from the unidentified tyrosine kinases in the lung tumor cells either. The altered regulation of Akt and MAPK in the tumor cells, however, further confirmed the existence of altered upstream kinases and/or phosphatases in the tumor cells.

Taken together, these results suggest PI 3K and MAPK-independent signaling pathway/pathways that may transmit the cell survival signals from the tyrosine kinases in the lung adenocarcinoma cells. This putative signaling pathway may share the same upstream tyrosine kinases with PI 3K and MAPK.

Several receptor tyrosine kinases have been reported to be expressed or overexpressed in lung adenocarcinoma cells (30, 31, 32, 33, 34, 35) . Blocking activation of these receptors inhibits cell growth (36 , 37) . To identify the tyrosine kinases responsible for the anoikis resistance, we analyzed the role of several known tyrosine kinases, including epidermal growth factor receptor, platelet-derived growth factor receptor, Src, and Abl in preventing the tumor cells from anoikis. Inhibition of some of these tyrosine kinases with their specific inhibitors increased anoikis in the noncancerous MDCK cells4 but failed to sensitize A549 lung adenocarcinoma cells to anoikis, indicating that these tyrosine kinases are not the ones that are responsible for the anoikis resistance of the tumor cells. Thus, the tyrosine kinases responsible for the anti-anoikis activity of the lung adenocarcinoma cells remain to be identified. Because the anti-anoikis signals generated by the tyrosine kinases are not transmitted through the PI 3K and the MAPK pathway, we propose that novel tyrosine kinase-dependent, PI 3K, and MAPK-independent signaling pathways may exist in the lung adenocarcinoma cells. Activation of these kinases and their downstream-signaling pathways circumvents the requirement of cell survival signals from the cell-matrix interactions and prevents the tumor cells from anoikis. Identification of the tyrosine kinases and the characterization of the new signaling pathway will be of therapeutic value in prevention of tumor cell proliferation and metastasis.


   FOOTNOTES
 
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.

1 Supported by NIH Grant R01 GM59678 from the National Institute of General Medical Sciences. Back

2 To whom requests for reprints should be addressed, at Pulmonary Center, Boston University Medical Center, 715 Albany Street, Boston, MA 02118. Phone: (617) 638-4466; Fax: (617) 536-8093; E-mail: QYU{at}LUNG.BUMC.BU.EDU Back

3 The abbreviations used are: FAK, focal adhesion kinase; PI 3K, phosphatidylinositol 3'-kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MDCK, Madin-Darby canine kidney; polyHEMA, polyhydroxyethylmethacrylate. Back

4 Unpublished data. Back

Received 3/29/00. Accepted 1/12/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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