Protein Kinase Cι Promotes Nicotine-induced Migration and Invasion of Cancer Cells via Phosphorylation of μ- and m-Calpains*

Nicotine is a major component in cigarette smoke that activates the growth-promoting pathways to facilitate the development of lung cancer. However, it is not clear whether nicotine affects cell motility to facilitate tumor metastasis. Here we discovered that nicotine potently induces phosphorylation of both μ- and m-calpains via activation of protein kinase Cι (PKCι), which is associated with accelerated migration and invasion of human lung cancer cells. Purified PKCι directly phosphorylates μ- and m-calpains in vitro. Overexpression of PKCι results in increased phosphorylation of both μ- and m-calpains in vivo. Nicotine also induces activation of c-Src, which is a known PKCι upstream kinase. Treatment of cells with the α7 nicotinic acetylcholine receptor inhibitor α-bungarotoxin can block nicotine-induced calpain phosphorylation with suppression of calpain activity, wound healing, cell migration, and invasion, indicating that nicotine-induced calpain phosphorylation occurs, at least in part, through a signaling pathway involving the upstream α7 nicotinic acetylcholine receptor. Intriguingly, depletion of PKCι by RNA interference suppresses nicotine-induced calpain phosphorylation, calpain activity, cell migration, and invasion, indicating that PKCι is a necessary component in nicotine-mediated cell motility signaling. Importantly, nicotine potently induces secretion of μ- and m-calpains from lung cancer cells into culture medium, which may have potential to cleave substrates in the extracellular matrix. These findings reveal a novel role for PKCι as a nicotine-activated, physiological calpain kinase that directly phosphorylates and activates calpains, leading to enhanced migration and invasion of human lung cancer cells.

Cigarette smoking, either active or passive, is the most important risk factor in the development of lung cancer. About 90% of male and 75-80% of female lung cancer deaths in the United States are caused by smoking (1)(2). Nicotine, a well known addictive component of tobacco, acts as a tumor promoter to facilitate the outgrowth of cells with genetic damage through the nicotinic acetylcholine receptor (nAChR) 2 -mediated signal transduction pathway (3)(4). Lung cancers, including small cell lung cancer (SCLC) and non-small lung cancer (NSCLC), are highly metastatic tumors with poor prognosis (5). Cigarette smoking has been demonstrated to promote tumor metastasis, but the mechanism(s) remains enigmatic (6 -7). The movement of cancer cells into tissue surrounding the tumor and the vasculature is the first step in the spread of metastatic cancers (8). Metastatic tumor cells have been observed to display more active motility, including migration and invasion, which appear to be a result of complex interplay between the numerous protein families participating in this process (9). Cell migration has been considered a required process during tumor cell invasion and metastasis (8,10). Tumor invasion involves cellular migration and interaction with the microenvironment at an ectopic site (11).
Because enhanced PKC activity is frequently found in cancer cells that show highly invasive and/or metastatic potential (12), cigarette smoke constituents (i.e. nicotine) may stimulate lung tumor metastasis by regulating PKC activity. PKC is a multigene family consisting of at least 11 distinct lipid-regulated protein-serine/threonine kinases that play pivotal roles in cell growth, apoptosis, differentiation, malignant transformation, and metastasis (13). This family can be divided into three subtypes: the classic isoforms (PKC␣, ␤I, ␤II, and ␥), which are Ca 2ϩ and diacylglycerol (DAG) dependent; the novel isoforms (PKC-␦, ⑀, , , and ), which are DAG dependent but Ca 2ϩ independent; and the atypical isoforms (PKC-and /), which possess only one zinc finger and lack the characteristic C2 domain and hence are insensitive to both Ca 2ϩ and DAG (14 -15). PKC isoenzymes exhibit distinct tissue distribution and play a distinct role in various cellular events, including cell survival, proliferation, tumorigenesis, tumor invasion, and metastasis (6 -7, 12, 16). For example, PKC, an atypical PKC isoform, presents predominantly in lung and brain (17), suggesting that PKC may play a critical role in regulating lung cancer development, including metastasis.
The mammalian calpains comprise 14 family members, of which -calpain (calpain 1) and m-calpain (calpain 2) are the best characterized calpain isoforms that are extensively expressed in both SCLC and NSCLC cells and are involved in the limited proteolysis of various focal adhesion structures and signaling enzymes to promote cell migration and invasion (18 -19). Bothand m-calpains function as heterodimeric enzymes composed of a unique, large catalytic subunit associated with a common, small regulatory subunit (calpain 4). Intriguingly, m-calpain can localize to integrin-associated adhesive structures, suggesting a potential mechanism by which m-calpain regulates cell migration (20). Studies utilizing pharmacological inhibitors and calpain knock-out cells indicate that calpain plays an important role in mediating the dynamic regulation of focal adhesions required for cell motility (21)(22)(23). In addition to Ca 2ϩ binding, our recent findings and those of others reveal that phosphorylation of calpain is another essential mech-anism for its activation (18, 24 -26). Here we discovered that nicotine, a major component in cigarette smoke, can induce phosphorylation and activation of bothand m-calpains through a novel signaling pathway involving ␣ 7 nAChR/c-Src/PKC that contributes to accelerated wound healing, migration, and invasion of human lung cancer cells.

EXPERIMENTAL PROCEDURES
Materials-Anti--calpain, m-calpain, PKC, c-Src, fluorescein isothiocyanate-conjugated anti-goat, and rhodamine-conjugated anti-rabbit IgG antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Nicotine and enolase were purchased from Sigma. Synthetic calpain substrate t-butoxy carbonyl-Leu-Met-chloromethylaminocoumarin (t-Boc-LM-CMAC) was obtained from Molecular Probes (Eugene, OR). The QCM TM chemotaxis 24-well colorimetric cell migration assay kit and cell invasion assay kit were purchased from Chemicon International, Inc. (Temecula, CA). Purified PKC was obtained from Pan-Vera (Madison, WI). PP2, ␣-bungarotoxin (␣-BTX), and c-Src enzyme were purchased from Calbiochem. The PKC/pAXneoRX construct was a kind gift from Dr. Alan P. Fields (27). All reagents used were purchased from commercial sources unless otherwise stated.
Metabolic Labeling, Immunoprecipitation, and Western Blot Analysis-Cells were cultured with 0.5% fetal bovine serum (FBS) medium overnight. Cells were washed three times with phosphate-free RPMI 1640 medium and metabolically labeled with [ 32 P]orthophosphoric acid for 90 min. After treatment with nicotine or inhibitor, cells were washed with ice-cold 1ϫ phosphate-buffered saline and lysed in detergent buffer.or m-calpain was immunoprecipitated using aor m-calpain antibody, respectively. The samples were subjected to 10% SDS-PAGE, transferred to a nitrocellulose membrane, and exposed to Kodak X-Omat film at Ϫ80°C for the time indicated. Phosphorylation ofor m-calpain was determined by autoradiography. The same filter was then probed by Western blot analysis using aor m-calpain antibody, respectively, and developed by using an ECL kit from Amersham Biosciences as described previously (18). Subcellular Fractionation-Subcellular fractionation was performed to isolate heavy membrane (HM), light membrane, cytosol, and nuclear membrane as previously described (28). Protein from each fraction was subjected to SDS-PAGE and analyzed by Western blotting using -calpain, m-calpain, and PKC antibodies. The purity of fractions was confirmed by assessing localization of fraction-specific proteins, including prohibitin (HM; Ref. 29 PKC Phosphorylatesor m-Calpain in Vitro--or m-calpain was immunoprecipitated from lysates of H1299 cells using an agarose-conjugatedor m-calpain antibody, respectively. The immunoprecipitatedor m-calpain was resuspended in a kinase assay buffer containing 50 mM Tris, pH 7.5,10 mM MgCl 2 , 0.5 mM EGTA, 0.1 mM CaCl 2, 10 M ATP, 40 g/ml phosphatidylserine, and 10 Ci of [␥-32 P]ATP. Purified, activated PKC enzyme (0.1 g) was added and incubated for 30 min at 30°C. The reaction was stopped by the addition of 2ϫ SDS sample buffer and boiling the sample for 5 min. The samples were analyzed by SDS-PAGE, and phosphorylation ofor m-calpain was determined by autoradiography.
Measurement of Intracellular c-Src Activity-Cells were stimulated with nicotine, harvested, and lysed in 0.5% Nonidet P-40 lysis buffer. The c-Src was immunoprecipitated from the lysates using a c-Src antibody. The complexes were washed three times with 500 l of lysis buffer and twice with c-Src kinase assay buffer (20 mM HEPES, pH 7.0, 10 mM MnCl 2 , 0.05% Triton X-100). The immune complex beads were suspended in 45 l of kinase assay buffer containing 1 g of acid-treated enolase as described (32). The kinase reaction was initiated by the addition of 2Ci of [␥-32 P]ATP, and the reaction mixture was incubated at 30°C for 10 min. The reaction was stopped by the addition of 50 l of 2ϫ SDS-PAGE sample buffer. Radiolabeled proteins were resolved by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and exposed to Kodak X-Omat film at Ϫ80°C for 24 h. The activity of c-Src was determined by autoradiography. The same filter was then probed by Western blot analysis using a c-Src antibody.
Purified c-Src Phosphorylates PKC in Vitro-PKC was immunoprecipitated from cell lysates of H1299 cells and incubated with purified c-Src enzyme (Calbiochem) in the c-Src kinase assay buffer containing 2 Ci of [␥-32 P]ATP at 30°C for 10 min as described above. Phosphorylation of PKC was determined by autoradiography.
Measurement of Intracellular PKC Activity-Cells were treated with nicotine for various times as indicated. PKC was immunoprecipitated from cell lysates with an agarose-conjugated PKC antibody. Immunoprecipitated PKC was washed and resuspended in 50 l of kinase assay buffer containing 50 mM Tris, pH 7.5,10 mM MgCl 2 , 0.5 mM EGTA, 0.1 mM CaCl 2, 10 M ATP, 40 g/ml phosphatidylserine, 10 g of histone-1, and 10 Ci of [␥-32 P]ATP. The reactions were incubated at room temperature for 30 min and terminated by addition of SDS sample buffer and boiling prior to SDS-polyacrylamide gel electrophoresis. The activity of PKC was determined by autoradiography.
Vector-based Gene Silencing of PKC by RNA Interference (RNAi)-The PKC DNA target sequence for siRNA design is AACTTCCTGAA-GAACATGCCA. This was determined by an Ambion siRNA Target Finder according to the human PKC cDNA sequence. The PKC-specific hairpin siRNA insert (sense-loop-antisense) was determined using a computerized insert design tool based on a target sequence following instructions from the Ambion website. Then, the oligonucleotide encoding the PKC-specific hairpin siRNA insert was synthesized and ligated into pSilencer TM 2.1-U6 hygro vector from Ambion (Austin, TX). The pSilencer TM 2.1-U6 hygro plasmids containing the PKC hairpin siRNA were transfected into H1299 cells using Lipofectamine TM -2000 according to the manufacturer's instructions. The stable clones persistently producing PKC siRNA were selected using hygromycin (0.8 mg/ml). The levels of PKC expression were analyzed by Western blot using a PKC antibody.
Detection of Calpain Activity in Living Cells-H1299 or H460 cells were plated at 50 -80% confluence in a glass chamber and incubated with 0.5% FBS medium for 24 h. The cells were then treated with nicotine or inhibitor in the presence of t-Boc-LM-CMAC (30 M) for 30 -60 min. Samples were observed under a fluorescent microscope (excitation 329 nm, emission 409 nm) as described (24 -25).
Cell Migration and Invasion Assay-Cells were treated with nicotine or inhibitor as indicated. Cell migration was assessed using a QCM TM 24-well colorimetric cell migration assay kit (Chemicon) following the manufacturer's instructions. This new technique does not require cell labeling, scraping, washing, or counting. Cells that migrated through the polycarbonate membrane were incubated with "Cell Stain Solution" and then subsequently extracted and detected on a standard microplate at 560 nm. Cell invasion was assessed using the Chemicon cell invasion assay kit. This assay was performed in an invasion chamber, which is a 24-well tissue plate with 12 cell culture inserts. The inserts contain an 8-m pore size polycarbonate membrane over which a thin layer of ECMatrix TM is dried. The extracellular matrix (ECM) layer occludes the membrane pores, blocking non-invasive cells from migrating through. Invasion cells migrate through the ECM layer and cling to the bottom of the polycarbonate membrane. The insert membrane with invaded cells on the bottom was placed in the wells with cell stain/dissociation solu-tion after incubation and reincubated for 30 min at 37°C. Absorbance was measured with a microplate reader at 560 nm. Each experiment was repeated three times, and data represent the mean Ϯ S.D. of three determinations.
Monolayer Wound Healing Assay-Cells were seeded into a six-well tissue culture dish and allowed to grow to 90% confluency in complete medium. Cell monolayers were wounded by a plastic tip (1 mm) that touched the plate as described (33). Wounded monolayers were then washed four times with medium to remove cell debris and incubated in 0.5% FBS medium in the absence or presence of nicotine (0.2 M) or inhibitor for various times up to 24 h. Cells were monitored under a microscope equipped with a camera (Deiss).
Quantitativeor m-Calpain ELISA Assay-The -calpain production in culture medium was measured using a -Calpain ELISA assay kit from Oncogene (San Diego, CA). This kit is designed to detect -calpain in tissue extracts, plasma, and serum. It uses a rabbit polyclonal antibody to the human -calpain large subunit immobilized on a microtiter plate to bind human -calpain. A highly purified, native human -calpain was used as a standard. The level of m-calpain in culture medium was also analyzed by ELISA using an m-calpain antibody from ARB Affinity Bioreagents (Golden, CO). Cells were cultured in RPMI1640 medium with 0.5% fetal bovine serum (FBS) and treated with nicotine or inhibitor as indicated. After centrifugation (1200 rpm), the resulting supernatant (culture medium) was collected. The level of -calpain or m-calpain in the medium was assessed by ELISA assay. The absorbance of the samples was analyzed using a microplate (ELISA) reader. Each experiment was repeated three times, and data represent the mean Ϯ S.D. of three determinations.

Nicotine Induces Phosphorylation and Activation ofand m-Calpains in Association with Accelerated Wound Healing, Migration, and
Invasion of Human Lung Cancer Cells--and m-Calpains are two major calpain family members that are widely expressed in both SCLC and NSCLC cells (Fig. 1A). Recently, reports indicate that epidermal growth factor-induced calpain phosphorylation enhances calpain activity in association with increased cell migration (24,34). To test whether nicotine can mimic a growth factor to regulate calpain, human lung cancer H1299 cells were metabolically labeled with [ 32 P]orthophosphoric acid and treated with nicotine (0.2 M) for various times. Phosphorylation ofand m-calpains was analyzed as described under "Experimental Procedures." Results reveal that nicotine potently stimulates phosphorylation of bothand m-calpains in human lung cancer cells within 60 -120 min (Fig. 1B). To test whether nicotine-induced calpain phosphorylation enhances its proteolytic activity, calpain activity in living cells was performed using t-butoxy carbonyl-Leu-Met-chloro- or m-Calpain was immunoprecipitated usingor m-calpain antibody, respectively. Phosphorylation ofor m-calpain was determined by autoradiography (upper). Western blot analysis using or m-calpain antibody was performed to confirm and quantifyor m-calpain protein (lower). C, H1299 cells were incubated with 0.5% FBS medium for 24 h. The cells were then treated with nicotine (0.2 M) in the presence of t-Boc-LM-CMAC (30 M) for 60 min. Calpain activity was analyzed by fluorescent microscopy. D, H1299 cells were seeded into a six-well tissue culture dish and allowed to grow to 90% confluency in complete medium. Cell monolayers were wounded by a plastic tip (1 mm) that touched the plate. Wounded monolayers were then washed four times with medium to remove cell debris and incubated in 0.5% FBS medium in the absence or presence of nicotine (0.2 M) for various times up to 24 h. Cells were monitored under a microscope equipped with a camera (Deiss). E, H1299 cells were treated with nicotine (0.2 M) for 24 h. Cell migration or invasion was measured using a QCM TM 24-well colorimetric cell migration assay kit or cell invasion kit, respectively. Each experiment was repeated three times, and data represent the mean Ϯ S.D. of three determinations.
methyl-aminocoumarin (t-Boc-LM-CMAC, a synthetic calpain substrate) as described (18,25). Cleavage of t-Boc-LM-CMAC by active calpains can induce retention of the chloromethylaminocoumarin portion of the molecule in the cells and can result in increased fluorescence (25). H1299 cells were treated with nicotine (0.2 M) in the presence of t-Boc-LM-CMAC for 60 min. Calpain activity was assessed by fluorescent microscopy. Results indicate that exposure of cells to nicotine enhances calpain activity (Fig. 1C). Recent reports have demonstrated that phosphorylation of calpain is able to increase its activity (18,25). Thus, nicotine-induced calpain activation may occur by a mechanism involving its phosphorylation. Other human lung cancer cell lines (i.e. H69, H82, H157, and H460 cells) that express various levels of endogenousand m-calpains were also tested, and similar results were obtained (data not shown).
Because calpains are involved in regulating cell migration and invasion (21,26), nicotine-induced phosphorylation and activation of calpains may promote migration and invasion of human lung cancer cells. To test this possibility, a wound-healing assay was employed as described under "Experimental Procedures." Compared with control cells, wound repair is significantly accelerated following treatment with nicotine ( Fig. 1D). Cell migration or invasion analysis using a QCM TM chemotaxis 24-well colorimetric cell migration or invasion assay kit reveals that nicotine significantly enhances both migration and invasion of human lung cancer cells (Fig. 1E). Collectively, these findings suggest that nicotine-stimulated migration and invasion of lung cancer cells may occur, at least in part, through phosphorylation and activation of calpains.
PKC Co-localizes and Interacts withand m-Calpains in Cytoplasm-PKC is extensively co-expressed withand m-calpains in both SCLC and NSCLC cells (Fig. 1A). To assess a potential direct role for PKC as a physiological calpain kinase, subcellular distribution of PKC and calpains was examined by subcellular fractionation assay. Results demonstrate that PKC is co-localized withor m-calpain in heavy membrane, light membrane, and cytosol in human lung cancer H1299 cells (Fig. 2A). To verify the purity of the subcellular fractions obtained, fraction-specific proteins were assessed by probing the same filters. Prohibitin, an exclusively mitochondrial protein (29), was detected only in the HM fraction. CPP32 (caspase 3), which is a cytosolic protease in growing cells (30), was detected exclusively in the cytosol, whereas proliferating cell nuclear antigen, which is a nuclear marker (31), was detected exclusively in the nuclear fraction ( Fig. 2A). These data reveal that each fraction obtained is highly pure without crosscontamination. To test whether nicotine stimulates an association between PKC and calpain, H1299 cells were treated with nicotine (0.2 M) for various times. A co-immunoprecipitation experiment was carried out using an agarose-conjugatedor m-calpain antibody. Calpain-associated PKC (i.e. bound PKC), -, or m-calpain were analyzed by Western blotting using PKC, -, or m-calpain antibody, respectively. Results reveal that treatment of cells with nicotine promotes PKC to associate with eitheror m-calpain in a time-dependent manner with maximal association between 30 -120 min (Fig. 2B). Thus, PKC may potentially function as a physiological kinase for bothand m-calpains to regulate their activities in human lung cancer cells.
Interestingly, the maximal association of PKC withor m-calpain happens earlier than their phosphorylation (30 versus 60 min, Figs. 1B and 2B). It is possible that PKC may first interact with calpains and then phosphorylate them as physiological substrates.
Nicotine Induces Activation of PKC, and PKC Phosphorylatesand m-Calpains in Vitro and in Vivo-PKC mRNA has been reported to predominantly express in brain and lung (17), suggesting that PKC may function as a major player in the development of human lung cancer. To test whether nicotine affects PKC activity, H1299 cells were treated with nicotine (0.2 M) for various times up to 180 min. PKC was immunoprecipitated using an agarose-conjugated PKC antibody. Activity of PKC was measured by an immune complex kinase assay using purified histone-1 as a substrate as described (35). The same filter was then probed by Western blot using a PKC antibody to quantify PKC protein. Results indicate that nicotine induces activation of PKC with a peak between 60 -120 min (Fig. 3A). Intriguingly, the peak time of PKC activity is consistent with the kinetics of nicotine-stimulated calpain phosphorylation (60 -120 min; Fig. 1B), suggesting a role of PKC in nicotine-induced calpain phosphorylation. To test whether activated PKC can directly phosphorylateand m-calpains,or m-calpain protein was immunoprecipitated from H1299 cells and incubated with purified, active PKC in a kinase assay buffer containing [␥-32 P]ATP as described under "Experimental Procedures." Importantly, active PKC directly phosphorylates bothand m-calpains in vitro (Fig. 3B). To determine whether PKC is a calpain kinase in vivo, a PKC/pAXneoRX expression construct or vector-only control was transfected into H460 cells that express relatively low levels of endogenous PKC. After transfection for 48 h, cells were metabolically labeled with [ 32 P]orthophosphoric acid and treated with nicotine (0.2 M) for 60 min. Results indicate that overexpression of PKC not only enhances nicotine-induced phosphorylation ofand m-calpains but also increases nicotine-stimulated migration and invasion of human lung cancer cells (Fig. 4). These findings provide both biochemical and genetic evidence thatand m-calpains are novel physiological PKC substrates in nicotine-activated cell migration and invasion signaling.

Nicotine Stimulates Activation of c-Src, Which Can Directly Phosphorylate and Activate PKC, and the Specific Src Inhibitor PP2 Suppresses Nicotine-stimulated Phosphorylation and Activation ofand m-Calpains in Association with Decreased Cell Migration and Invasion-Our
data show that nicotine induces activation of PKC, which can directly phosphorylate calpains (Figs. 3 and 4). However, the upstream protein kinase(s) for PKC activation remains unclear. PKC is insensitive to Ca 2ϩ due to the absence of the calcium-binding domain (16). Thus, nicotine-induced activation of PKC may occur through a calcium-independent mechanism. Because c-Src has been reported to directly induce tyrosine phosphorylation of PKC at tyrosine 256, 271, and 325 sites along with activation of enzyme activity (32), and c-Src is ubiquitously expressed in both SCLC and NSCLC cells (Fig. 1A), we postulate that nicotine may stimulate c-Src activity to phosphorylate and activate PKC. To test this, H1299 cells expressing endogenous c-Src were treated with nicotine for various times, followed by immunoprecipitation of c-Src and measurement of its activity by an immune complex kinase assay with acid-treated enolase as a substrate. Results reveal that nicotine potently enhances c-Src activity in a time-dependent manner (Fig. 5A). To test whether c-Src can directly phosphorylate endogenous PKC, PKC protein was immunoprecipitated from H1299 cells and incubated with purified, active c-Src in a kinase assay buffer containing [␥-32 P]ATP as described under "Experimental Procedures." Results indicate that active c-Src directly phosphorylates PKC in vitro (Fig. 5B). These findings suggest that c-Src may function as an upstream PKC kinase in nicotine-stimulated cell migration and invasion signaling. To pharmacologically test this, H1299 cells were treated with nicotine in the absence or presence of increasing concentrations of the Src-specific inhibitor PP2 (32). Results show that PP2 not only inhibits nicotineinduced calpain phosphorylation and activation but also potently suppresses cell migration and invasion (Fig. 6). Collectively, these findings suggest that nicotine-induced migration and invasion of human lung cancer cells may occur by a mechanism involving the c-Src/PKC/calpain signal pathway.

The ␣ 7 nAChR-specific Inhibitor ␣-BTX Inhibits Nicotine-induced Phosphorylation and Activation ofand m-Calpains in Association with Suppression of Migration, Invasion, and Wound Healing of Human
Lung Cancer Cells-␣-BTX has been identified as the site-selective antagonist for ␣ 7 nAChR (36). Because ␣ 7 nAChR plays an important role in lung cancer cell signaling (36) and nicotine is a site-selective, high affinity agonist for the ␣ 7 nAchR (36 -37), we tested whether ␣-BTX affects nicotine-induced phosphorylation ofand m-calpains in H1299 lung cancer cells. Results indicate that ␣-BTX potently blocks nicotine-induced phosphorylation ofand m-calpains in association with decreased calpain activity, cell migration, and invasion (Fig. 7,  A-C). Importantly, ␣-BTX potently inhibits nicotine-stimulated wound healing (Fig. 7D). These findings suggest that ␣ 7 nAChR functions as the upstream receptor in nicotine-induced calpain phosphorylation as well as migration and invasion of human lung cancer cells.
Depletion of PKC Suppresses Nicotine-induced Calpain Phosphorylation, Migration, and Invasion of Human Lung Cancer Cells-Our findings suggest that PKC functions as a nicotine-activated calpain kinase

. Overexpression of PKC results in increased phosphorylation ofand m-calpains and accelerated cell migration and invasion.
A, PKC/pAXneoRX construct was overexpressed in H460 cells. Expression levels of PKC were determined by Western blot using a PKC antibody. B, H460 cells overexpressing PKC or vector control cells were metabolically labeled with [ 32 P]orthophosphoric acid and treated with nicotine (0.2 M) for 60 min. Phosphorylation ofor m-calpain was analyzed as described in Fig. 1B. C, H460 cells overexpressing PKC or vector control cells were treated with nicotine (0.2 M) for 24 h. Cell migration or invasion was measured using a QCM TM 24-well colorimetric cell migration assay kit or cell invasion kit, respectively. Each experiment was repeated three times, and data represent the mean Ϯ S.D. of three determinations. in human lung cancer cells (Figs. 1-4). To test whether PKC is essential for nicotine-stimulated calpain phosphorylation, a vector-based stable gene silencing approach was employed for specific depletion of PKC from human lung cancer cells. The pSilencer TM 2.1-U6 hygro plasmids bearing the PKC hairpin siRNA insert were transfected into H1299 cells. The stable clones persistently producing PKC siRNA were selected using hygromycin. Results indicate that cells expressing PKC siRNA display Ͼ90% reduction of PKC protein expression (Fig. 8A). Importantly, specific disruption of PKC expression by RNAi blocks nicotine-induced phosphorylation ofand m-calpains in association with suppression of nicotine-stimulated calpain activity, cell migration, invasion, and wound healing (Fig. 8). These findings indicate that PKC is an essential kinase for nicotine-induced calpain phosphorylation, cell migration, and invasion.
Nicotine Enhances Secretion of Calpains from Human Lung Cancer Cells, Which Could Be Blocked by ␣-BTX or PP2-Cell motility is regulated by both intrinsic and extrinsic cellular properties (38). Because cell migration and invasion are involved in disassembly of adhesion complexes associated with the ECM, it is possible that nicotine-induced phosphorylation ofand m-calpains may regulate their release from the cytoplasm to the ECM. To test this hypothesis, H1299 cells were treated with nicotine (0.2 M) in the absence or presence of increasing concentrations of ␣-BTX or PP2 for 24 h. Theor m-calpain production in culture medium was measured using a -or m-calpain ELISA assay as described under "Experimental Procedures." Results reveal that nicotine potently enhances secretion of bothand m-calpains within 24 h (Fig. 9). Intriguingly, the ␣ 7 nAChR inhibitor ␣-BTX and the Srcspecific inhibitor PP2 not only suppress nicotine-induced calpain phosphorylation (Figs. 6 and 7) but also significantly block secretion of both and m-calpains (Fig. 9). These findings suggest that nicotine-induced calpain phosphorylation may affect calpain secretion.

DISCUSSION
Cigarette smoke is by far the number one cause of lung cancer, and ϳ90% of lung cancer occurs in smokers or former smokers (5). Tumor progression to the invasive and metastatic states dramatically enhances the mortality of cancer. Lung cancer has a very poor prognosis because of its high metastatic potential. The liver and adrenal gland have been found to be frequent sites of spread from lung cancer (5). Rational therapeutic interventions will only be possible when we understand the molecular mechanisms governing cellular behavior underlying this transformation. For invasion, a subpopulation of tumor cells must recognize, modify, and migrate through the ECM barrier and then proliferate in the adjacent but ectopic locale (39). Therefore, the proteolytic ability of the cell is a key factor in the processes of cell migration and invasion. Calpain has been reported to be a positive regulator of cell migration and invasion because it localizes to focal adhesions and cleaves many focal adhesion-related proteins in the ECM, including integrin receptors, focal adhesion kinase, and talin, etc. (40). Intriguingly, calpain activity is significantly elevated in various tumor cells when compared with nonmalignant cells (41)(42). A recent in vivo study indicates that antisense-mediated suppression of m-calpain inhibited invasion of prostate carcinoma cells (11). Thus, elevated calpain activity may enhance motility of tumor cells, which may potentially promote tumor metastasis.
Growing evidence indicates that cigarette smoking facilitates the spread of cancer in the body in various types of cancer, including human lung and breast cancers (6 -7). However, the intracellular signal mechanism by which cigarette smoking promotes metastasis and/or development of lung cancer remains elusive. Here we discovered that nicotine, a major component in cigarette smoke, can stimulate phosphorylation ofand m-calpains with increased proteolytic activity in human lung cancer cells (Fig. 1). Functionally, treatment of lung cancer cells with nicotine (0.2 M) results in accelerated wound healing and enhanced cell migration and invasion (Fig. 1, D and E). Phosphorylation of calpain has been demonstrated to positively regulate its proteolytic activity (18,(25)(26). Thus, nicotine-stimulated cell motility signaling may occur in a mechanism through phosphorylation ofand m-calpains that enhances their activities. Because nicotine exists at high concentrations (0.09ϳ1 M) in the blood of smoking patients (44), the concentration of nicotine (0.2 M) in our experiments falls within the range found in the blood of smoking patients.
PKC is an atypical PKC isoform, and Northern blot analysis, using the full-length PKC cDNA as a probe, revealed that the PKC transcript presents predominantly in lung and brain (17). Consistently, our data show that PKC is ubiquitously expressed in both human SCLC and NSCLC cells (Fig. 1A). It is possible that PKC may play a pivotal role in regulating lung cancer metastasis. It is known that PKC is implicated in tumor invasion and metastasis (12), but the downstream substrate for this effect is not clear. Evidence reported here suggests that PKC functions as a calpain kinase because PKC co-localizes and interacts withand m-calpains in the cytoplasm and directly phosphorylates eitheror m-calpain in vitro, indicating its potential, direct role as a calpain kinase (Figs. 2 and 3). Confirmation of PKC as a physiological calpain kinase was obtained in vivo from results of transfection studies demonstrating that PKC, when overexpressed in H460 cells, resulted in enhanced phosphorylation ofand m-calpains, which are consistent with in vitro results (Fig. 4). Importantly, specific knockdown of PKC expression by RNAi can significantly inhibit nicotine-stimulated calpain phosphorylation and activity in association with decreased migration and invasion of H1299 lung cancer cells (Fig. 8). These findings strongly indicate that PKC functions as a physiological nicotine-activated calpain kinase that positively regulates calpain activity through phosphorylation, leading to migration and invasion of human lung cancer cells.
PKC belongs to an atypical PKC isoenzyme category that differs significantly from other PKC family members in their regulatory domain in that it lacks both the calcium-binding domain and one of the two zinc finger motifs required for DAG binding (16). These domain variations result in a different requirement for activation. Because PKC is insensitive to both Ca 2ϩ and DAG (16), other mechanisms, for example, phosphorylation or protein-protein interaction, may be required for PKC activation. Recent studies reveal that c-Src not only induces tyrosine phosphorylation of PKC but also directly binds to PKC, which leads to its activation (32). Because nicotine can induce activation of c-Src, which could then in turn phosphorylate PKC (Fig. 5), these findings strongly suggest that c-Src most likely functions as a nicotineactivated upstream PKC kinase. nAChRs are cationic channels whose opening is controlled by acetylcholine and nicotinic receptor agonists. The ␣ 7 nAChR is expressed in normal human small airway epithelial cells, SCLC and NSLC cells (36 -37, 45). ␣-BTX has been identified as the site-selective antagonist for ␣ 7 , but not ␣ 3 ␤ 4 , nAChRs (36,46,47). Because ␣-BTX potently blocks both nicotine-induced phosphorylation and activation ofand m-calpains in association with inhibition of wound healing, cell migration, and invasion ( Fig. 7), this indicates that nicotine-induced cell migration and invasion may occur through activation of the ␣ 7 nAChR signal transduction pathway involving ␣ 7 nAChR/c-Src/PKC/calpains in lung cancer cells. Importantly, ␣-BTX may have potential clinical relevance in strategies designed to restrain tumor invasion and metastasis through this novel mechanism in patients with lung cancer.
In addition to PKC, our previous report and others have demonstrated that mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 can function as nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-or epidermal growth factoractivated calpain kinases (18,24). The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-specific inhibitor PD98059 can partially inhibit NNK-induced calpain phosphorylation as well as cell migration and invasion (18). Thus, various types of calpain kinases may cooperatively regulate calpain activity through its phosphorylation. However, PKC may play a more extensive and/or more important role in nicotine-stimulated cell migration and invasion signaling because depletion of PKC by RNAi not only blocks nicotine-induced calpain phosphorylation but also significantly suppresses migration and invasion of human lung cancer cells (Fig. 8).
Calpain was generally believed to exist and function only in the cytoplasm. Recent studies indicate that m-calpain has also been detected in the extracellular space of tissue that results from active secretion rather than cell destruction (43). We have previously demonstrated that treatment of SCLC H69 cells with nitrosamine 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone enhances secretion ofand m-calpains by a mechanism through vesicles (18). Here we found that nicotine can also stimulate secretion ofand m-calpains from NSCLC H1299 cells into culture medium (Fig. 9). These secreted extracellular calpains may have the potential to cleave ECM, leading to increased lung cancer cell invasion and/or metastasis. Importantly, treatment of cells with ␣-BTX or PP2 inhibits nicotine-induced calpain phosphorylation in association with decreased calpain secretion (Figs. 6, 7, and 9), suggesting that nicotine-induced calpain phosphorylation may be involved in regulation of its secretion. Further studies will be required to demonstrate this possibility.
In summary, our findings have identified PKC as a novel nicotine-activated protein kinase that directly phosphorylates and activatesand m-calpains. Thus, nicotine-stimulated migration and invasion of human lung cancer cells may occur, at least in part, through a novel signaling pathway involving ␣ 7 nAChR/c-Src/PKC/calpains. Nicotineinduced calpain phosphorylation through PKC enhances both activity and secretion ofand m-calpains, which may lead to cleavage of focal adhesion-related proteins and promote lung cancer metastasis. These findings may have potential clinical relevance for restraint of lung cancer metastasis by blocking upstream of nicotine-activated calpain signaling pathways.