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Experimental Therapeutics, Molecular Targets and Chemical Biology |
1 Division of Experimental Therapeutics, 2 Department of Medical Oncology and Hematology, Ontario Cancer Institute/Princess Margaret Hospital, and 3 Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; and 4 Kinetek Pharmaceuticals, Inc., Vancouver, British Columbia, Canada
Requests for reprints: David W. Hedley, Department of Medical Oncology and Hematology, Princess Margaret Hospital, 610 University Avenue Toronto, Ontario, Canada M5G 2M9. Phone: 416-946-2911; Fax: 416-946-6546; E-mail: david.hedley{at}uhn.on.ca.
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Abstract |
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Key Words: protein kinase B • Akt • mammalian target of rapamycin • signal transducers and activators of transcription-3 • forkhead transcription factors
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Introduction |
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Recently, the integrin-linked kinase (ILK) has been identified as an important member of the PI3K-PKB pathway. ILK is an ankyrin repeat–containing serine-threonine protein kinase that mediates a diversity of cell functions by coupling integrins and growth factors to cascades of downstream signaling events. This protein is widely expressed throughout the body, with high levels of expression in the pancreas, and the cardiac and skeletal muscles (8, 9). Overexpression of ILK in mammary and intestinal epithelial cells leads to the stimulation of anchorage-independent cell growth, cell cycle progression, and constitutive up-regulation of cyclins D and A expression and tumorigenicity in nude mice (10, 11). Furthermore, elevated ILK expression and activity have been correlated with melanoma (12) as well as with cancers of the breast, prostate, brain, colon, stomach, and ovary (13–18). Hence, ILK seems to be important in carcinogenesis.
ILK is a downstream substrate of PI3K and an important upstream kinase for the regulation of PKB/Akt (9). For PKB to become fully activated, it must be recruited to the plasma membrane and become phosphorylated at both Thr308 and Ser473 sites (19). Thr308 is phosphorylated by phosphoinositide-dependent kinase 1 (PDK1) and recent studies have shown that in certain circumstances, ILK is involved in the phosphorylation of Ser473 site. Fully activated PKB can then mediate diverse signaling events through the phosphorylation of glycogen synthase kinase-3ß (GSK-3ß), forkhead in rhabdomyosarcoma (FKHR) transcription factor, and the mammalian target of rapamycin (mTOR) pathways. GSK-3ß has been proposed to be phosphorylated and inhibited directly by ILK or indirectly through the fully active phosphorylated form of PKB (20). The inhibition of GSK-3ß results in the activation of ß-catenin/lymphoid enhancing factor 1 and activator protein 1 transcription factors as well as the stabilization and accumulation of cyclin D1 in the nucleus leading to activated transcription, cell cycle progression, and proliferation (10, 20). The phosphorylation of FKHR by PKB, on the other hand, is followed by nuclear exclusion and inhibition in the expression of FOXO-regulated genes such as the cell cycle inhibitor p27Kip1 and proapoptotic molecules FasL and Bim (21, 22) . Additionally, the activation of the mTOR pathway by PKB phosphorylates signal transducers and activators of transcription 3 (Stat3) Ser727, resulting in transcription of genes involved in cell differentiation, proliferation, and apoptosis (23); 4E-BP1, thereby releasing transcription factor eIF4E for DNA binding and ultimately increasing protein synthesis (24); and S6K1 and its downstream substrate S6 ribosomal protein, leading to increased cell growth (24, 25). Recently, it has been reported that both S6K1 and 4E-BP1/eIF-4E pathways are required for and independently mediate mTOR-dependent G1 phase progression (26). Therefore, the activation of PKB by ILK has the potential for extensive downstream effects in the regulation of cell signaling pathways. Consequently, deregulation of the PI3K/ILK/PKB pathway might play a major role in cancer development and growth.
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Materials and Methods |
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Drug Preparation. ILK inhibitor QLT0254 was obtained from Kinetek Pharmaceuticals, Inc. (recently acquired by QLT Inc., Vancouver, British Columbia, Canada). QLT0254 has been shown to inhibit the kinase activity of ILK in cell-free assay at 185 nmol/L and preliminary experiments suggest that it has 
100-fold selectivity over other kinases tested under similar conditions, including CDK2, CDK5, CK2, CSK, ERK1, GSK3ß, LCK, PIM1, PKA, DNA-PK, and PKB/Akt (QLT, Inc.).5 Of those tested, CDK1, PKC, and FYN show the greatest inhibition by QLT0254 but the selectivity window is still close to 100. The drug was prepared as a 10 mg/mL suspension by adding 5% Tween 80 in milli-Q water to the powder, vortexing vigorously for 2 minutes, sonicating for 1 minute, and heating at 65°C for 2 minutes. This procedure (vortexing, sonicating, and heating) was done twice. The drug was prepared fresh every week and stored at room temperature. Gemcitabine was obtained from Eli Lilly & Co. (Indianapolis, IN), and prepared as a 20 mg/mL solution in sterile PBS.
Treatment Protocols. In the time course experiment, five groups of three randomly assigned, tumor-bearing OCIP#4 SCID mice were treated with 200 mg/kg QLT0254 i.p. (except for the 0 h group) before sacrificing and harvesting the tumors at 0, 2, 4, 8, and 24 hours.
In the tumor growth inhibition experiment, the QLT0254 dose was reduced from 200 to 100 mg/kg. Two groups of eight randomly assigned, tumor-bearing OCIP#4 SCID mice were given QLT0254 (100 mg/kg) or the vehicle control [5% Tween 80 in double-distilled water (ddH2O)]. All treatments were given by i.p. injections, five times consecutively in a week, for a total of 3 weeks. At the end of the third week, the animals were sacrificed and the tumors were harvested and weighed after removal of nontumoral tissues.
Next, the single-dose acute combination therapy experiment was replicated once to include a total of 24 tumor-bearing animals with 6 animals randomly assigned to one of four groups: (a) drug-vehicle controls (PBS and 5% Tween 80 in ddH2O, i.p.), (b) gemcitabine (80 mg/kg, i.p.), (c) QLT0254 (150 mg/kg, i.p.), and (d) gemcitabine and QLT0254 (80 and 150 mg/kg respectively, i.p.). For group d, gemcitabine was given at 0 hour followed by QLT0254 at 24 hours. A similar schedule was used in the other groups except that the drug not included in the treatment regimen was replaced with the corresponding vehicle. All mice were killed 48 hours after beginning the experiment.
Last, the 4-week combination therapy experiment consisted of the same sample size and treatment groups as the previous single-dose acute combination therapy experiment; however, in this experiment both gemcitabine and QLT0254 were administered i.p. twice a week, with gemcitabine on Mondays and Thursdays and the ILK inhibitor on Tuesdays and Fridays for the combination group. Again, other groups followed a similar schedule with the exception that the drug not included in the regimen was substituted with the corresponding vehicle. After 4 weeks of treatment, the animals were killed and the tumors were harvested and weighed immediately.
Depending on the subsequent analyses, all harvested tumors were either cut into pieces and fixed in 10% formalin for immunohistochemistry, snap-frozen in OCT (Miles, Inc., Elkhart, IN) in liquid nitrogen for immunofluorescence or treated with lysis buffer for Western blots.
Western Blot Analysis. Minced tumor pieces were homogenized in 1 mL lysis buffer [50 mmol/L HEPES (pH 8.0), 10% glycerol, 1% Triton X-100, 150 mmol/L NaCl, 1 mmol/L EDTA, 1.5 mmol/L MgCl2, 100 mmol/L NaF, 10 mmol/L NaP2O7·H2O, 1 mmol/L NaVO4] containing protease inhibitor cocktail tablets (Roche Canada, Mississauga, Ontario, Canada) for 1 hour on ice. Homogenates were clarified by centrifuging at 14,000 rpm at 4°C for 15 minutes. Samples were then heated in sample buffer for 10 minutes at 95°C, run on 10% SDS-polyacrylamide gels, and transferred to nitrocellulose membranes using the Mini Trans-Blot Electrophoresis Transfer Cell (Bio-Rad Laboratories, Mississauga, Ontario, Canada). Membrane blots were blocked for 1 hour at room temperature with 10% nonfat milk in TBS with 1% Tween 20 and then incubated overnight at 4°C with the following primary antibodies: PKB (1:500, Cell Signaling Technology, Beverly MA), ILK1 (1:500), P-PKB/Akt Ser473 (1:1000), P-PKB Thr308 (1:500), P-PDK1 Ser241 (1:500), P-GSK3ß Ser9 (1:1000), P-p44/42 MAP Kinase (1:1000), P-FKHR Ser256 (1:500), P-p70S6K Thr389 (1:1000), P-S6 ribosomal protein Ser235/236 and Ser240/244 (1:1000), P-4E-BP1 Ser65 (1:500), P-Stat3 Ser727 (1:500), P-Stat3 Tyr705 (1:500), and ß-actin (1:25,000, Abcam, Cambridge, MA). The blots were then probed with either the anti-rabbit polyclonal or anti-mouse monoclonal secondary antibody containing horseradish peroxidase (Amersham Biosciences, Baie d'Urfe, Quebec, Canada) at 1:1,000 the next morning for 1 hour at room temperature before exposing to ECL (Amersham Biosciences) per manufacturer's instructions. Blots were either incubated once with a primary antibody or stripped once, blocked, and reprobed with a different antibody overnight at 4°C. Densitometric analyses for P-PKB/Akt Ser473 were done using densitometry and ImageQuant v.3.0 (Molecular Dynamics, Sunnyvale, CA).
Immunohistochemistry. Paraffin-embedded sections of OCIP#4 tumor tissues from the time course experiment were stained with H&E, monoclonal ILK antibody (1:200, Upstate Biotechnology, Lake Placid, NY), phosphospecific polyclonal antibodies PKB/Akt Ser473 (1:400) and GSK-3ß Ser9 (1:500), and developed with a streptavidin/biotin–peroxidase method.
Indirect Immunofluorescence. Indirect immunofluorescence was used for the frozen sections obtained from the tumor growth inhibition and the acute combination therapy experiments. Serial sections (5-µm-thick) were cut from OCT-embedded frozen tissue and fixed in 2% formaldehyde for 20 minutes. One section was stained with H&E for the selection of the tumoral areas with transmitted light microscopy. The other sections were stained with the following: (a) CD31 (1:500, BD Biosciences, Franklin Lakes, NJ) and labeled with the Cy3-conjugated rat anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA) for detection of vasculature, (b) proliferation marker Ki-67 and the early apoptosis marker, cleaved caspase-3 (1:100, DAKO, Glostrup, Denmark; 1:50, Cell Signaling Technology) with secondary antibodies Alexa 488 and a Cy5-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch) respectively. Controls for nonspecific background were used by staining sections with secondary antibody alone. All sections were counterstained with DNA specific dye 4',6-diamidino-2-phenylindole to outline the nuclear area.
Computerized Image Analysis. Composite digital images of the entire H&E sections were acquired as described previously (28). On these H&E images, the total viable tumor area was selected for analysis of the double-labeled Ki-67/cleaved caspase-3 sections, whereas the viable tumor and stroma of the viable tumor were selected for analysis of the CD31 marker.
The selected tumoral areas of the CD31 and the Ki-67/cleaved caspase-3 sections were examined using a Microcomputer Imaging Device image analysis system (Imaging Research Inc., St. Catharines, Ontario, Canada) equipped with a cooled charged coupled device camera mounted on an epifluorescence microscope. For each tumor, the analyzed area was obtained by tiling an entire section at 20x magnification (2.24 mm2) to create composite images of individual fields as previously described (29).
To examine Ki-67 staining (nuclear), the nuclear image obtained with 4',6-diamidino-2-phenylindole was binarized to form masks, which were then overlaid onto the Ki-67 fluorescence image to capture Ki-67 staining only within the nucleus. A size criterion (mean value of positive Ki-67 nuclei) was selected to count the number of positive objects. The percentage of nuclear Ki-67 was then acquired by normalizing the number of positive nuclear Ki-67 objects over the number of nuclei stained with 4',6-diamidino-2-phenylindole and multiplying by 100. Cleaved caspase-3 was measured as the percentage of positively stained area that is the proportional area (total cleaved caspase-3 positive area/total viable tumoral area) expressed as a percentage. The CD31 images were also binarized and measured as the number of positive objects per tumoral area for microvessels (5 µm2 > all vessels < 150 µm2) and average proportional area for total vascular area (all vessels > 20 µm2).
Statistics. All values are presented as mean ± SE. Comparisons between tw groups (control versus QLT0254) were achieved using the two-tailed Student's t test, whereas comparisons between four treatment groups in the single-dose acute combination therapy experiment and the 4-week combination therapy experiment were done using Kruskal-Wallis one-way ANOVA on ranks and subsequently the Student-Newman-Keuls method for all pairwise multiple comparisons between the groups. The criterion for statistical significance is P < 0.05.
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Figure 3D shows the Western blots of the two well-characterized substrates downstream of the mTOR pathway. The two isoforms p70S6K and p85S6K, together termed S6K1, were detected in these OCIP#4 lysates in the phosphorylated form, with the nuclear p85S6K protein having much higher basal levels at 0 hour after very short exposure to film than the cytoplasmic p70S6K isoform, which appeared as a faint band after longer exposure. Treatment with QLT0254 decreased phosphorylated p85S6K at the mTOR-regulated Thr412 site after 2 hours of treatment and this effect remained until slight recovery at 24 hours. The P-p70S6K isoform also showed dephosphorylation at 2 hours, but this effect was not as pronounced.
S6 ribosomal protein is the primary substrate phosphorylated by P-S6K1 (Fig. 1). As shown in Fig. 3D, P-S6 was considerably dephosphorylated at residues Ser240/244, with slight recovery from 4 to 24 hours. Conversely, Ser235/236, another residue of S6, also becomes dephosphorylated but to a lesser extent than the Ser240/244 site. At Ser235/236, dephosphorylation occurs slightly after 2 hours but phosphorylation returned after 8 hours. Recently, it was shown that in the absence of S6K1 and S6K2 (an isoform of S6K1) activity, a MAPK pathway cooperates in the regulation of S6 phosphorylation at Ser235/236 (34). Therefore, the weaker dephosphorylation pattern of S6 at Ser235/236 than Ser240/244 may be due to signaling from the MAPK pathway in the absence of S6K1 and perhaps S6K2, but we did not examine the protein levels of S6K2 in this study. This conforms to the finding that P-ERK remained unaffected with treatment (Fig. 3C). Another important substrate, 4E-BP1, undergoes inhibitory phosphorylation by mTOR. Inhibition of ILK with QLT0254 led to an almost complete ablation of this protein after 2 hours and recovery at 4 hours.
Stat3, another target of mTOR, is phosphorylated at Ser727 (Fig. 1) and QLT0254 treatment led to a dephosphorylation after 2 hours with recovery after 8 hours (Fig. 3E). The tyrosine site of Stat3, on the other hand, is phosphorylated by cytokine receptors through JAK or directly through activated receptor and nonreceptor tyrosine kinases at the plasma membrane before its dimerization and translocation to the nucleus for phosphorylation of the serine site by mTOR (23) or by ERK (35). This tyrosine site, however, becomes dephosphorylated slightly by 4 to 8 hours, with more dephosphorylation at 8 hours before reappearance of the activated form at 24 hours (Fig. 3E). PKB protein levels show equal loading of samples. Similar results were obtained from tumors of three different animals killed at each time point. Overall, these results show that a single i.p. dose of QLT0254 at 200 mg/kg is able to dephosphorylate PKB, FKHR, S6K1, S6 ribosomal protein, 4E-BP1, and Stat3 without affecting the protein levels of PKB, ILK, P-PDK1, P-GSK-3ß, and P-ERK in the OCIP#4 human orthotopic pancreatic tumors (Fig. 1).
Tumor Growth Inhibition after 3-Week QLT0254 Treatment as a Single Agent. There were three deaths in the control group before the end of the 3-week treatment because of large tumor. QLT0254 was well tolerated in the tumor-bearing SCID mice. All animals in this group were alive and exhibited a healthier appearance than the control animals. On average, the control mice lost 1.31 g from the initial weight of 24.66 g, whereas QLT0254-treated mice lost only 0.47 g from the initial 25.47 g but the difference in weights between the two groups at the termination of the study were not statistically significant. Because of the location of the tumor in the orthotopic pancreas site, the tumor size could not be measured until at the end of the experiment when the animals were sacrificed. Consequently, at the end of the 3-week treatment, orthotopic tumors were dissected free of surrounding normal tissues and weighed. As shown in Fig. 4A, treatment with 100 mg/kg of QLT0254 five times a week for a total of 3 weeks significantly inhibited tumor growth by 2-fold compared with vehicle-treated controls (P = 0.004).
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Effects of 3 Weeks Administration of QLT0254 on Proliferation and Apoptosis. Indirect immunofluorescence staining for nuclear Ki-67 in the tumor growth inhibition tumors was used. Figure 4D displays a bar graph of the percentage of nuclear Ki-67 in the vehicle control and QLT0254-treated tumors. Although on average, QLT0254-treated tumors revealed less Ki-67 staining than the control tumors, the difference between the means was statistically insignificant (P = 0.323).
Figure 4E compares the percentage of positively stained cleaved caspase-3 area between the control and QLT0254-treated tumors. The data showed no statistically significant effect of QLT0254 in inducing apoptosis in QLT0254-treated tumors compared with vehicle controls (P = 0.369). The same sections were also stained for terminal deoxynucleotidyl transferase–mediated nick end labeling and revealed similar results (data not shown). Nonetheless, there was a trend toward more apoptosis and less proliferation in QLT0254-treated tumors and more proliferation and less apoptosis in the control tumors.
Single-dose Acute Proliferative and Apoptotic Effects of Combination Therapy. Figure 5A compares the percentage of nuclear Ki-67 between the drug-vehicle control group and the xenografts treated with a single dose of 150 mg/kg QLT0254 i.p. for 24 hours. In general, there was a decrease in Ki-67 labeling in QLT0254-treated mice but this effect was not statistically significant (P = 0.374).
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Tumor Growth Inhibition after 4-Week Combination Therapy. Figure 6 compares the tumor weights of OCIP#4 animals treated with i.p. injections of the vehicle control, 80 mg/kg of gemcitabine twice a week, 100 mg/kg of QLT0254 twice a week, or in combination with 80 mg/kg of gemcitabine on Mondays and Thursdays and 100 mg/kg of QLT0254 on Tuesdays and Fridays for 4 weeks. There were significant differences between the control versus the gemcitabine alone group, control versus combined treatment group, gemcitabine alone versus QLT0254 alone group, and QLT0254 alone versus combined treatment group (P < 0.001). Nevertheless, because these tumors were relatively sensitive to gemcitabine alone, there was no statistically significant enhancement in tumor reduction when treatment was combined with QLT0254 (P > 0.05), although there is a suggestion that the addition of QLT0254 to gemcitabine resulted in a further reduction in tumor size. Furthermore, the actual mean tumor weight of the combined treated group is less than that shown in Fig. 6 because histologic examination of the H&E sections in this group revealed microscopic tumors surrounded by substantial amounts of nontumoral tissue that could not be distinguished from the tumor during the naked eye dissection.
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Discussion |
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The small molecule inhibitor QLT0254 acts to inhibit the catalytic activity of ILK, which in turn can block the phosphorylation of GSK-3ß and PKB/Akt Ser473, leading to the incomplete activation of PKB/Akt. Structurally related ILK inhibitors have been tested in phosphatase and tensin homologue–mutated prostate cancer cell lines PC-3 and LNCaP, resulting in the suppression of phosphorylated PKB/Akt at Ser473 but not Thr308 (30). In the present study, we found >80% inhibition of P-PKB/Akt Ser473 levels following a single dose of QLT0254 in vivo, with recovery over 24 hours consistent with the pharmacokinetic profile of the compound. However, we also observed slight dephosphorylation in PKB Thr308 protein, possibly due to minor nonspecific effects of the compound at the relatively high dose of 200 mg/kg (close to maximum tolerated dose) because P-PDK1 was not affected with treatment. Regardless, the inhibitory effects were much more profound at the Ser473 residue. Altogether, these results showed the efficacy of QLT0254 in suppressing ILK and further support the notion that ILK can lead to the phosphorylation of PKB/Akt Ser473.
Tan et al. (36) have shown that other analogues of QLT0254, KP-SD-1, and KP-SD-2 can suppress LS-180 human colon cancer cell and s.c. xenograft tumor growth. In the colon cancer xenografts, the ILK inhibitors blocked PKB/Akt Ser473 and GSK-3ß phosphorylation (36). Administration of QLT0254 for 3 weeks to our primary orthotopic pancreatic cancer xenografts also resulted in tumor growth inhibition and suppression of PKB/Akt phosphorylation but with no effects in phosphorylated GSK-3ß. The failure to suppress phosphorylation of GSK-3ß despite the inhibition of its upstream kinases ILK and PKB by QLT0254 might be attributed to signaling of GSK-3ß by other upstream regulators, such as Dishevelled, GBP/FRAT1, PKC
, and PP2A from the Wnt signaling pathway (37), as well as PKA (38, 39) and SGK (40). This sustained phosphorylation of GSK-3ß and the consequent proliferation may be one factor leading to the insignificant decrease in Ki-67 labeling in QLT0254-treated tumors compared with controls in both single dose as well as the 3-week tumor inhibition studies.
In addition to down-regulation of P-PKB/Akt Ser473, continuous treatment with QLT0254 for 3 weeks resulted in significant tumor growth delay. The suppression of P-PKB/Akt Ser473 in these tumors might potentially result in the initiation of apoptosis and cell cycle arrest via the dephosphorylation of FKHR and Stat3 (21, 23) or via effects on protein synthesis, cell growth, and proliferation downstream of mTOR through inactivation of S6K1 and activation of 4E-BP1 (24–26). Tan et al. (41) have recently reported that QLT0254 can also inhibit tumor angiogenesis in PC3 prostate cancer xenografts, but we did not see this effect in the OCIP#4 model. Although the proliferation and apoptosis effects of QLT0254 on OCIP#4 were not statistically significant, there was a tendency both toward more apoptosis and less proliferation in QLT0254-treated tumors than the controls, suggesting that these combined effects might be responsible for the significant tumor growth inhibition.
We cannot exclude the possibility that in addition to effects on the malignant cell population in OCIP#4 tumors, growth inhibition by QLT0254 is also mediated by effects on the tumor stroma because this expresses higher levels of ILK. Although the suppression of P-PKB Ser473 in the cancer cells suggests that these are in fact the more important drug target, further work is needed to investigate the role of ILK in tumor/stroma interactions in vivo.
It has been proposed that the activation of the PI3K/PKB pathway can produce drug resistance in pancreatic cancers, although other signaling mechanisms might be involved in some cases (42). Our group and others have shown that PI3K inhibitors are able to sensitize pancreatic cancer cells to gemcitabine and that combined treatment is more effective in xenograft models (3, 4, 7) . In accordance with these findings, we found that a single dose of QLT0254 enhanced gemcitabine-induced apoptosis in OCIP#4 xenografts. There was also a suggestion that combined treatment twice weekly was more effective than single agents. However, these data are difficult to interpret because of the relatively high sensitivity of OCIP#4 to gemcitabine. Moreover, potential for ILK inhibition to enhance gemcitabine effects in pancreatic cancers requires further testing using different xenograft models.
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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 James Ho, Trudey Nicklee, Jennifer Woo, and Pinjiang Cao for their technical assistance, and Diana Birle for helpful discussions.
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Footnotes |
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5 A. Stewart, personal communication. 
Received 8/13/04. Revised 11/13/04. Accepted 12/ 6/04.
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A. A. Troussard, P. C. McDonald, E. D. Wederell, N. M. Mawji, N. R. Filipenko, K. A. Gelmon, J. E. Kucab, S. E. Dunn, J. T. Emerman, M. B. Bally, et al. Preferential Dependence of Breast Cancer Cells versus Normal Cells on Integrin-Linked Kinase for Protein Kinase B/Akt Activation and Cell Survival Cancer Res., January 1, 2006; 66(1): 393 - 403. [Abstract] [Full Text] [PDF]
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 M. Saleem, S. Kaur, M.-H. Kweon, V. M. Adhami, F. Afaq, and H. Mukhtar Lupeol, a fruit and vegetable based triterpene, induces apoptotic death of human pancreatic adenocarcinoma cells via inhibition of Ras signaling pathway Carcinogenesis, November 1, 2005; 26(11): 1956 - 1964. [Abstract] [Full Text] [PDF]
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M. N. Younes, S. Kim, O. G. Yigitbasi, M. Mandal, S. A. Jasser, Y. Dakak Yazici, B. A. Schiff, A. El-Naggar, B. N. Bekele, G. B. Mills, et al. Integrin-linked kinase is a potential therapeutic target for anaplastic thyroid cancer Mol. Cancer Ther., August 1, 2005; 4(8): 1146 - 1156. [Abstract] [Full Text] [PDF]
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