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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
Departments of 1 Experimental Therapeutics, 2 Pathology, and 3 Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center; 4 Cancer Biology Program, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas
Requests for reprints: Kapil Mehta, Department of Experimental Therapeutics, Unit 362, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-8140; Fax: 713-745-4167; E-mail: kmehta{at}mdanderson.org.
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Introduction |
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Cytotoxic chemotherapy and surgery are used as palliative treatment for PDAC patients. 5-Fluorouracil and gemcitabine are the most commonly used drugs to treat pancreatic cancer (2–5), but PDAC exhibits intrinsic resistance to these drugs (6). Therefore, understanding the novel pathways and proteins that contribute to the development of resistance to anticancer therapies may help to identify novel targets for PDAC treatment.
Recently, we reported that drug-resistant and metastatic breast cancer and melanoma cells expressed elevated levels of tissue transglutaminase (TG2; refs. 7–10). Importantly, the increased TG2 expression was associated with an increased resistance to chemotherapeutic drugs and other apoptosis-inducing stimuli. Inhibition of TG2 by small interfering RNA (siRNA) reversed the sensitivity of drug-resistant MCF-7 breast cancer cells to doxorubicin and rendered them sensitive to serum withdrawal–induced apoptosis (6, 7).
TG2 is ubiquitous and most diverse member of the transglutaminase family of enzymes (11). In addition to catalyzing Ca2+-dependent post-translational modification of proteins, it can catalyze Ca2+-independent hydrolysis of guanosine 5'-triphosphate and adenosine 5'-triphosphate. The ability of TG2 to hydrolyze guanosine 5'-triphosphate enables it to serve as a signaling molecule, leading to the activation of a cytoplasmic target, phospholipase C (12, 13). Moreover, the high affinity of TG2 for fibronectin may play a role in promoting integrin-mediated cell adhesion (14) and cell migration functions (15, 16). Similarly, TG2 has been shown to activate RhoA and mitogen-activated protein kinase pathways (MAPK; ref. 17), which control key signaling pathways that impinge on the invasive and metastatic behavior of malignant cells. Several reports have documented elevated expression of TG2 in various cancer types (7–11, 18, 19).
In this report, we examined the constitutive expression of TG2 in 75 PDAC tumors and 12 PDAC cell lines. The results were correlated with various molecular markers and clinicopathologic data. We showed that elevated TG2 expression was associated with gemcitibine resistance and invasive phenotypes in pancreatic cancers. Furthermore, we examined the molecular mechanisms by which TG2 overexpression lead to gemcitabine resistance and increased invasive potential in PDAC cell lines using adenovirus overexpression system, siRNA knockdown, and confocal microscopy. We showed, for the first time, that TG2 can interact with focal adhesion kinase (FAK) and result in its activation; this function was independent of the transamidation activity of TG2. Therefore, we concluded that elevated TG2 expression in PDAC cells can contribute to the development of drug resistance and invasive phenotypes by activation of the FAK/phosphatidylinositol 3-kinase (PI3K)/AKT pathway.
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Materials and Methods |
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The PDAC cell lines AsPC-1, MiaPaCa-2, Panc-1, HPAF-II, Hs766T, Capan-1, Capan-2, and BxPC-3 were provided by Dr. Shrikanth A. Reddy (The University of Texas M. D. Anderson Cancer Center, Houston, TX). Panc-28, Panc-02-3, Panc-48, and Su8686 cell lines were provided by Dr. Paul J. Chiao (The University of Texas M. D. Anderson Cancer Center). All cell lines were maintained in the log phase of cell growth by being cultured in RPMI 1640 or DMEM/F-12 supplemented with FCS (10%, v/v), Normocin (0.1 mg/mL), L-glutamine (2 mmol/L), and HEPES (10 mmol/L; U.S. Biochemical, Cleveland, OH) at 37°C in a CO2 incubator. The immortalized pancreatic ductal epithelium cell line (E6E7) was provided by Dr. Ming Tsao (Ontario Cancer Institute, Toronto, Ontario, Canada) and cultured in keratinocyte serum-free medium supplemented with bovine pituitary extracts, human EGF, and FCS.
TG2 enzymatic activity. Cells at 70% to 80% confluence were washed in PBS and collected in a minimal volume of the lysis buffer [20 mmol/L Tris-HCl (pH 7.4) containing 1 mmol/L EDTA, 150 mmol/L NaCl, 14 mmol/L 2-mercaptoethanol, 1 mmol/L phenylmethylsulphonyl fluoride]. Cells were then lysed in the same buffer by probe sonication and assayed for TG2 activity by determining the Ca2+-dependent incorporation of [3H]putrescine (specific activity, 14.3 Ci/mmol; Amersham Pharmacia, San Francisco, CA) into dimethylcasein as described previously (20). The enzyme activity was expressed as nanomoles of putrescine incorporated per milligram of total cell protein.
Western blotting. Cells were serum starved for 12 to 16 hours before the experiment. The whole-cell lysate (60 µg) or fractions thereof (prepared using the Biovision cell fractionation kit according to the manufacturer's protocol) were fractionated by 4% to 15% gradient SDS-PAGE. After SDS-PAGE, the proteins were electrotransferred onto nitrocellulose membranes, blotted with each antibody, and detected with enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ). Some of the membranes were stripped using Restore stripping buffer (Pierce, Rockford, IL) for reprobing with another antibody. The protein bands obtained were quantified using AlphaEase FC (Fluor Chem 8900) software from Alpha Innotech (San Leandro, CA).
Immunoprecipitation. Cells were lysed in a minimum volume of Tris-HCl buffer [50 mmol/L (pH 8)] containing 150 mmol/L NaCl and 1% NP40 and precleared by incubation with 50 µL Trueblot anti-mouse Ig IP beads for 1 hour at 4°C. The pellet was discarded, and the supernatant was subjected to immunoprecipitation: 200 µg cell lysate was incubated with 2 µg of specific antibody for 1 hour at 4°C. Twenty microliters of Trueblot anti-mouse Ig IP beads was added, and the pellet was further incubated on a rotating device overnight at 4°C. The pellet was then washed four times in ice-cold lysis buffer. The supernatant was discarded, and the pellet was resuspended in 50 µL of the sample buffer. The samples were fractionated by SDS-PAGE and analyzed by immunoblotting and autoradiography.
Cytotoxicity. The number of viable cells remaining after the appropriate treatment was determined by measuring their ability to reduce 3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) into a soluble formazan in accordance with the manufacturer's instructions. Logarithmically growing cells were plated at 2 x 103 per well in 96-well plates and allowed to adhere overnight. They were then cultured alone or with 0.1 nmol/L to 100 µmol/L gemcitabine for 24, 24, or 72 hours. Each experiment was repeated thrice in triplicate. The concentration of gemcitabine required to inhibit proliferation by 50% (IC50) was calculated. Under identical conditions, the cells were trypsinized, and the cell viability was determined with a trypan blue exclusion test to confirm the MTS results.
In vitro invasion. The invasive potential of PDAC cell lines was studied in vitro by determining the number of cells that invaded through Matrigel-coated Transwell polycarbonate membrane inserts as described previously (21). In brief, Transwell inserts with a pore size of 12 µm were coated with 0.78 mg/mL Matrigel in serum-free medium. Cells were recovered by trypsinization, washed, and resuspended in serum-free medium, and 0.5 mL of the cell suspension (0.5 x 106 cells) was added to duplicate wells. After incubation for 48 hours, the cells that passed through the filter were stained using a Hema-3 stain kit (Fisher Scientific, Houston, TX). The cells in 10 random fields were counted under a microscope.
Wild-type and C277S mutant TG2 adenovirus. An adenovirus containing wild-type (WT; TG2wt) or C277S mutant TG2 (TG2m) cDNA was kindly provided by Dr. Ugra Singh (The Texas A&M University System Health Science Center, Temple, TX). In brief, TG2 cDNA cloned in pcDNA3.1 vector was first subcloned in a pshuttle 2 vector and then in a BD adenoX adenoviral vector. HEK293 cells were transfected with recombinant adenoviral plasmid for packaging of adenovirus particles. The adenovirus was purified on a CsCl2 gradient and used at 25 multiplicities of infection (MOI). Cells infected with lacZ adenovirus served as the control.
TG2 down-regulation by siRNA. Two TG2 siRNA sequences were designed and purchased from Qiagen (Germantown, MD). A sequence that did not have homology to any human mRNA (as determined by a BLAST search) served as a control, whereas two sequences were designed to target TG2 mRNA, siRNA1 (target sequence, 5'-AAGGCCCGTTTTCCACTAAGA-3') and siRNA2 (target sequence, 5'-AAGGGCGAACCACCTGAACAA-3'). For transfection, 2 x 105 cells were plated in each well of six-well plates and allowed to adhere for 24 hours. On the day of transfection, 30 µL RNAiFect transfection reagent (Qiagen) was added to 5 µL siRNA (1 µg/µL) in a 65-µL culture medium to give a final volume of 100 µL. The siRNA/transfection reagent mixture was incubated at ambient temperature for 15 minutes and added uniformly to plates in serum-containing medium. After 48 hours of transfection, cells were recovered and used for appropriate determinations. The transfection efficiency was determined by transfecting cells in a parallel well with fluorescent siRNA and determining fluorescence uptake under the microscope.
Confocal microscopy. To determine the colocalization of phosphorylated FAK (pFAK) and TG2, cells were grown on glass coverslips and fixed in 4% paraformaldehyde for 20 minutes at ambient temperature. Fixed cells were then incubated with the primary antibodies overnight, washed with PBS, and incubated again with secondary antibodies conjugated with either Alexa 546 (red) or 488 (green; Molecular Probes, Eugene, OR). The DNA dye Topro-3 (Molecular Probes) was used to costain the nuclei (blue). Cells treated with secondary antibodies alone were used as controls. A confocal scanning analysis of the cells was done with a Zeiss laser scanning confocal microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY) or an Olympus FluoView 300 confocal microscope (Center Valley, PA) in accordance with established methods, using sequential laser excitation to minimize the fluorescent emission bleed through. Each section was examined for the presence of each stain at two excitations (546 and 488 nm), and the data were compared pixel by pixel. Each image represented z sections at the same cellular level and magnification; a three-dimensional reconstructed image was used to visualize the whole sample. Merging red and green showed colocalization of two proteins, giving a yellow color.
Immunohistochemistry. Samples used in this study were from patients with primary PDAC who underwent initial pancreaticoduodenectomy at our institution between 1990 and 2004. None of these patients received preoperative chemotherapy or radiation before surgery. A total of 75 such patients were identified for whom tissue samples and follow-up information were available. Patients' clinicopathologic data were collected and follow-up data were updated through December 31, 2005 by reviewing medical records and the U.S. Social Security Index. The use of archival paraffin-embedded tissue blocks and chart reviews were approved by the Institutional Review Board Committee of M. D. Anderson Cancer Center. Tissue microarrays were constructed using formalin-fixed, paraffin-embedded archival tissue blocks from these 75 PDACs using the method as described previously (22). The H&E-stained slides were reviewed and screened for most representative areas of the tumor and their matched paraffin blocks were retrieved. Each tumor and adjacent benign pancreatic tissue were sampled in duplicate with 1.0-mm tissue cores to include two tissue cores from the tumor and two tissue cores from the paired benign pancreatic tissue. In addition, nine different human pancreatic adenocarcinoma cell lines were also included to serve as controls.
TG2 expression levels in tumor samples were evaluated by an indirect immunoperoxidase procedure (avidin-biotin complex method Elite; Vector Laboratories, Burlingame, CA). In brief, antigen retrieval was done by treating the tissue samples in a steamer for 30 minutes. Antibody (0.5 µg/mL) against TG2 (CUB7401, Neomarkers) overlaying the tissue section was incubated at 4°C for 16 hours. The secondary antibody incubation was done at ambient temperature for 1 hour. Mayer's hematoxylin nuclear stain was used as a counterstain. Immunostaining results were evaluated and scored independently by a pathologist and laboratory personnel. TG2 expression in tumor cells was categorized as negative (low or weak cytoplasmic staining) or positive (diffuse moderate to strong cytoplasmic staining).
Statistical analysis. The clinicopathologic and follow-up data were correlated with TG2 expression. The statistical analysis was done using Fisher's exact test and Student's t test with SPSS software (version 12 for Windows; SPSS, Chicago, IL) and a P < 0.05 was considered significant.
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Results |
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To further confirm the observation that TG2 overexpression increased the invasive potential and confer resistance to gemcitabine in PDAC cells, we overexpressed the WT (TG2wt) and catalytically inactive (TG2m) mutant of TG2 in BxPC-3 cells. TG2m lacks transamidation activity because of a point mutation in the active site cysteine residue (C277S). The results shown in Fig. 3A and B suggested that overexpression of either TG2wt and TG2m strongly promotes the invasive potential of BxPC-3 cells. Moreover, BxPC-3 cells infected with TG2wt adenoviral construct became significantly more resistant to gemcitabine-induced cytotoxicity when compared with the adenovirus alone-infected cells (IC50, 0.1 µmol/L; P = 0.03; data not shown). Together, these results suggested that overexpression of TG2 contributes not only to gemcitabine resistance but also to the invasive potential of PDAC cells, which was independent of its transamidation activity.
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To further delineate the relationship between TG2 and FAK, we overexpressed TG2 by infecting BxPC-3 cells with 25 MOI of an adenovirus-containing TG2 WT (TG2wt) construct. As expected, TG2 expression in infected BxPC-3 cells increased by 10- to 12-fold over the basal level as determined by Western blotting and confocal microscopy (Fig. 4A and C ). Overexpression of TG2 was associated with a parallel increase in the enzyme activity (Fig. 4B) and constitutive activation of FAK in BxPC-3 cells. The infection of cells with adenovirus vector alone had no effect on TG2 expression or FAK activation. Interestingly, overexpression of TG2m, which lacks the enzyme activity, also resulted in an increase in FAK activation similar to that induced by TG2wt (Fig. 4A and B). These results suggested that TG2-mediated activation of FAK occurs independent of its transamidation activity. This was further supported by confocal microscopy data (Fig. 4C). Interestingly, the TG2-mediated activation of FAK was associated with an increase in the colocalization of TG2 with pFAK at the focal adhesion points (Fig. 4C).
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Down-regulation of TG2 inhibits FAK activation. To further determine the role of TG2 in FAK activation, we used siRNA approach to down-regulate the endogenous expression in Panc-28 cells. As reported previously (8, 9), siRNA1 and siRNA2 caused 80% to 90% inhibition of endogenous TG2 protein expression in Panc-28 cells (Fig. 5A ). The down-regulation of TG2 expression by siRNA resulted in inactivation of FAK (Fig. 5A). However, transfection of Panc-28 cells under similar conditions with control siRNA did not alter either TG2 expression or FAK activation (Fig. 5A). Down-regulation of TG2 in Panc-28 cells also resulted in marked morphologic changes by light microscopy (Fig. 5B): the cells appeared more cylindrical in shape and segregated than were untreated controls. More importantly, the inhibition of endogenous TG2 by siRNA induced massive cytoplasmic vacuolization after 48 hours of transfection (Fig. 5B), and the cells eventually died. In addition, the down-regulation of endogenous TG2 was associated with a profound inhibition of Panc-28 cells to invade through Matrigel-Transwell membranes (data not shown). These results clearly showed that TG2 expression plays a critical role in the constitutive activation of FAK and its downstream functions and that TG2 expression is critical for the survival of Panc-28 cells.
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80% of the total TG2 protein was present in the cytosolic fraction and 15% was in the membrane. Notably, the fraction of TG2 bound with FAK was higher in the membrane fraction (77%) than in the cytosolic fraction (33%; Fig. 6B). Because activated FAK is mainly localized in the cytoplasmic membrane, we speculated that TG2 in the membrane was associated with the phosphorylated form of FAK. This contention was supported by the results of immunoprecipitation of Panc-28 cell extracts with an anti-pFAK (pY397) antibody. As shown in Fig. 6C, TG2 is indeed associated with pFAK. The results of a confocal microscopy analysis (Fig. 6D) further supported the colocalization of TG2 with pFAK, particularly at the focal adhesion points. The colocalization of TG2 and pFAK at focal points was quantified in 40 cells, showing a mean number of 6.2 overlaps at focal points per cell. These results show, for the first time, that there is a direct association between TG2 and the signaling molecule FAK and that this association plays an important role in FAK activation. TG2 expression in PDAC tumor samples. To further evaluate the significance of our in vitro observations that TG2 expression results in gemcitabine resistance and invasive potential, we examined the expression levels of TG2 in a human tissue microarray containing 75 PDAC tissue samples. We found high basal levels of TG2 expression in 42 of the 75 (56%) tumor samples studied (Fig. 7A ). However, only 24 of 70 (34%) cases showed cytoplasmic staining in benign pancreatic ducts. TG2 expression was significantly higher in PDACs compared with normal ducts (P = 0.012). No staining was observed in sections treated with the isotypic control IgG instead of the primary anti-TG2 antibody (data not shown). High TG2 expression in tumor samples was significantly associated with nodal metastasis [32 of 48 node-positive tumor samples were TG2 positive versus 10 of 27 node-negative tumor samples; odds ratio (OR), 3.400; P = 0.017], lymphovascular invasion (33 of 51 lymphovascular invasive tumors were TG2 positive versus 9 of 24 noninvasive tumors; OR, 3.055; P = 0.045), and clinical stage (30 of 45 stage IIb PDACs were TG2 positive versus 10 of 26 stage IIa tumors; OR, 3.200; P = 0.027). These data further supported our observations that TG2 overexpression increases the invasive potential of PDAC cell lines. A Kaplan-Meier estimate of survival in relation to TG2 expression showed no significant difference in TG2-positive patients (n = 42) and TG2-negative patients (n = 33; P = 0.159; Fig. 7B).
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Discussion |
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Previously, we found that constitutive expression of TG2 in drug-resistant and metastatic breast cancer (8) and late-stage melanoma (9) was up-regulated. Importantly, down-regulation of TG2 expression was associated with the increased sensitivity of cancer cells to apoptotic stimuli and reversal of the drug-resistant phenotype (8). Han and Park (23) reported similar results in the drug-resistant lung cancer cell line PC-14/ADR. These authors showed that the acquisition of multidrug resistance in PC-14 cells was associated with a 10- to 15-fold increase in TG2 expression and that inhibition of TG2 expression by TG2-specific antisense or ribozyme rendered the cells sensitive not only to multidrug resistance–related drugs but also to other anticancer drugs.
In this study, we determined the constitutive TG2 expression status of PDAC tumors and tumor cell lines. Of the 12 PDAC cell lines tested, 10 expressed high levels of TG2 protein, and two expressed moderate levels (Fig. 1). Importantly, 56% of the PDAC tumor samples showed elevated levels of TG2, which was associated with nodal metastasis, lymphovascular invasion, and late clinical stage. In cell lines, TG2 expression was associated with resistance to gemcitabine (Fig. 2B), a commonly used drug for the treatment of pancreatic cancer. Similarly, high TG2 expression was associated with invasive behavior (Fig. 2C). Thus, ectopic expression of TG2 in BxPC-3 cells rendered the cells highly invasive (Fig. 4D) and resistant to gemcitabine and, conversely, the inhibition of TG2 by siRNA significantly attenuated the invasive potential of Panc-28 cells.
To delineate the possible mechanism by which TG2 could promote chemoresistance and invasive potential in PDAC cells, we studied the relationship between TG2 and FAK. TG2 has been shown to promote FAK activation via its ability to promote the integrin-mediated attachment of cells to fibronectin (8, 14), a major component of the extracellular matrix in tumor microenvironments. FAK is a nonreceptor cytoplasmic protein tyrosine kinase that provides signaling and scaffolding functions at sites of integrin adhesion. Ample evidence supports the role of FAK in cell adhesion, cell migration, and cell cycle progression (24). Increased FAK expression is frequently associated with malignant or metastatic disease and poor patient prognosis (24–30). FAK expression is increased in various tumors, such as those of the breast, colon, thyroid, head and neck, ovaries, liver, esophagus, and pancreas (24–30). The activated form of FAK constitutes tyrosine phosphorylation at the COOH terminus (Y397). This results in the phosphorylation of other tyrosine residues (Y397, Y407, Y576, Y577, Y861, and Y925), leading to the activation of several downstream signaling pathways, such as RAS/ERK, PI3K/AKT, and Crk/Dock180/Rac (ref. 24 and references therein).
The data presented here suggest a novel pathway that could lead to the constitutive activation of FAK in pancreatic and possibly other aggressive cancer cells. TG2 was closely associated with FAK protein. The interaction of TG2 with FAK was associated with FAK autophosphorylation (pY397; Fig. 4). Indeed, ectopic expression of TG2 in BxPC-3 cells resulted in enhanced FAK activation (Fig. 4C) and FAK colocalization with TG2 in the focal points (Fig. 6D). Conversely, the inhibition of TG2 by siRNA inhibited FAK activation in Panc-28 cells (Fig. 5A). The major fraction of activated FAK was localized in the membrane in complex with TG2 (Fig. 6B). The ability of TG2 to associate with FAK and subsequent phosphorylation of FAK were independent of TG2 transamidation activity, as suggested by the observation that the expression of catalytically inactive point-mutated (C277S) TG2 could result in a similar association and activation of FAK (Fig. 4A). Together, our results suggest a role for TG2 in FAK autophosphorylation that is mediated by a direct association between the two proteins. How the association of TG2 with FAK affects autophosphorylation of FAK remains a matter of speculation. Indeed, it is well known that the recruitment of FAK and its association with the cytoplasmic tails of integrins result in its autophosphorylation (24). Similarly, it is likely that the conformational changes induced in FAK in response to its interaction with TG2 can result in its autophosphorylation. Alternatively, the association of TG2 with pFAK might protect it from the action of endogenous phosphatases, such as PTEN (31). We are currently investigating these and other possibilities to determine the mechanisms that underlie the phosphorylation of FAK in response to its interaction with TG2.
Furthermore, TG2-mediated FAK activation was associated with the activation of PI3K/AKT pathway but had no effect on the RAS/MEK/ERK pathway (Fig. 4A). These observations are supported by an earlier report by Kang et al. (32) who showed that overexpression of TG2 in human chronic myelogenous leukemia (K562) cells was associated with activation of the PI3K/AKT signaling pathway (33). The significance of the PI3K/AKT pathway in conferring an aggressive phenotype to cancer cells has been well established (32, 34, 35). Indeed, our earlier studies with drug-resistant and metastatic breast cancer cells revealed that down-regulation of TG2 expression renders the cells sensitive not only to anticancer drugs but also to stress-induced apoptosis (8, 9). In the present study, the inhibition of endogenous TG2 by siRNA induced a massive accumulation of cytoplasmic vacuoles (Fig. 5B) that eventually led to the death of Panc-28 cells via autophagy.5
Overall, the results of our study show that elevated expression of TG2 in PDAC tumor cells and cell lines contributes to increased cell survival and invasive functions. One mechanism that likely contributes to these effects is the ability of TG2 to associate with and activate FAK, resulting in the activation of its downstream prosurvival PI3K/AKT signaling pathway. In conclusion, our results suggest that TG2 could be an attractive target for anticancer therapeutics, and they provide a strong rationale for developing TG2 inhibitors to target the disruption of TG2-FAK protein-protein interactions.
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Acknowledgments |
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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.
We thank Ann M. Sutton for editorial help.
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Footnotes |
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Received 6/29/06. Revised 8/10/06. Accepted 9/ 7/06.
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