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Tumor Biology |
Departments of Oncology [L. B. M., R. B., J. S., J. D., N. A., R. G., T. B., A. C., C. M-C., J. P-S., R. J.], Pathology [L. B. M., J. D., N. A., R. Fal., R. Fai., C. M-C., S. L., R. J.], and Surgery [J. S., J. P-S.], H. Lee Moffitt Cancer Center and Research Institute, University of South Florida College of Medicine, Tampa, Florida 33612, and Antisense Drug Discovery, Isis Pharmaceuticals, Carlsbad, California 92008 [J. K.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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STATs4 were identified originally as central players in signaling pathways involved in mediating responses to IFNs and other cytokines (reviewed in Refs. 6, 7, 8 ). On phosphorylation of STAT monomers by tyrosine kinases, the monomers dimerize, translocate to the nucleus, and bind to specific promoter sequences, thereby inducing gene expression. STAT proteins have essential functions in signaling pathways critical to normal cellular processes including immune function, development, differentiation, proliferation, and survival (9, 10, 11, 12) . One of the cytokines known to activate Stat3 is IL-6. IL-6 regulates a wide variety of biological responses and stimulates or inhibits cellular growth in a cell type-dependent manner (13 , 14) . Furthermore, activation of Stat3 by IL-6 has a critical role in the pathogenesis of multiple myeloma by preventing apoptosis (15) . In addition, IL-6 has been suggested to participate in malignant progression of prostate cancer (16) .
Earlier studies have reported that constitutive activation of one STAT family member, Stat3, occurs in fibroblasts stably transformed by the Src oncoprotein and other oncoproteins that activate tyrosine kinase signaling pathways (17, 18, 19, 20) . More recent studies have demonstrated that Stat3-mediated expression of cellular genes is required for Src-induced oncogenic transformation (21, 22, 23) . In addition, constitutive STAT activation occurs frequently in a variety of human tumor cell lines and primary human tumors including leukemias, lymphomas, multiple myeloma, head and neck cancer, and breast cancer (24, 25, 26, 27) . Moreover, a constitutively activated mutant of Stat3 is sufficient to induce some properties of transformed cells, including tumorigenicity (28) . Collectively, these findings provide evidence that signaling by Stat3 protein participates in regulating the processes of cell growth and survival during oncogenesis.
Because cytokines including IL-6 have been implicated in prostate cancer, we examined Stat3 signaling in primary tumor specimens from prostate cancer patients and in human prostate cancer cell lines. Using both a biochemical Stat3 DNA-binding assay and an immunohistochemical phospho-Stat3 assay, we demonstrate that Stat3 is constitutively activated with high frequency in prostate adenocarcinomas compared with matched adjacent nontumor prostate tissues. In addition, we used antisense Stat3 oligonucleotides to block Stat3 signaling and to evaluate the biological role of activated Stat3 in model prostate cancer cell lines. Results indicate that activated Stat3 transduces signals that are required for growth and survival of human prostate cancer cells. Our findings implicate constitutive activation of Stat3 in malignant progression of prostate cancer and suggest that Stat3 represents a promising molecular target for novel prostate cancer therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pathology Evaluation.
Prostatectomy specimens were sent immediately to the pathology laboratory on ice with a label indicating the time of excision (to monitor processing time from excision to snap-freezing). Every effort was made to minimize processing time to less than 15 min because it was extremely important to snap-freeze tumor specimens in liquid nitrogen as soon as possible to preserve the activation state of STAT proteins in tumor cells. The specimen was cut at 5-mm intervals, and areas of normal and tumor tissue were identified grossly with the guidance of biopsy results obtained before prostatectomy. The frozen sections were evaluated by the pathologists (J. D., L. B. M.) to confirm the diagnosis of both normal and tumor tissue. This approach assured reliable comparison of biochemical data in tumoral and nontumoral tissue. Excess tissue not required for pathological examination of the specimen was obtained for the purposes of this study. Informed consents were signed by all patients to allow the use of their tissues in these experiments as part of a clinical protocol approved by the local Institutional Review Board. Tissue adjacent to (mirror-image area) the confirmed tumor or normal tissue by frozen section evaluation was snap-frozen immediately in liquid nitrogen and sent to the Moffitt Cancer Center Tumor Bank for future STAT studies. The remainder of the prostatectomy specimen was fixed in formalin and processed for routine histological examination and immunohistochemical analysis.
Cell Lines and Culture Conditions.
The human prostate carcinoma cell lines LNCaP, DU145, and PC3 were obtained from the American Type Culture Collection (Rockville, MD) and were grown in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FBS. IL-6 stimulation experiments were performed using 10 ng/ml IL-6 in serum-free medium for the indicated times.
Nuclear Extract Preparation and EMSA.
For STAT analysis by EMSA, the frozen tissue specimens were placed in a prechilled mortar and pestle on liquid nitrogen, and the tissue was pulverized in the frozen state. Nuclear extracts were prepared as described previously (17
, 20)
from primary tissue specimens with approximately 106 cells by high-salt extraction into 30–70 µl buffer [20 mM HEPES (pH 7.9), 420 mM NaCl, 1 mM EDTA, 20% glycerol, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 µM aprotinin, 1 µM leupeptin, and 1 µM antipain] producing about 30–90 µg of total protein. For EMSA, 5 µg of total extracted protein was used for each lane. EMSA was performed using a 32P-radiolabeled oligonucleotide probe containing a hSIE derived from the c-fos gene promoter (sense strand, 5'-AGCTTCATTTCCCGTAAATCCCTA-3') that binds activated Stat1 and Stat3 proteins (17)
. Protein-DNA complexes were resolved by nondenaturing PAGE and detected by autoradiography. For controls, STAT activity was determined in matched specimens obtained from nontumor tissues adjacent to the tumor, and also in NIH3T3 cells transformed by the Src oncoprotein (17)
as an internal reference standard. Thus, levels of STAT activation were compared in nontumor and tumor tissues of the same patient, and normalization to the Src internal reference standard allowed comparison across different EMSA gels. Quantification of Stat3 activation levels was performed using ImageQuant software analysis of scanned EMSA gel bands. Anti-Stat1 and Stat3 polyclonal antibodies (Santa Cruz Biotechnology) were used to identify specific STAT family members. For use in supershift assays, 1 µl of the STAT antibodies was incubated with nuclear extracts for 20 min at room temperature before the addition of radiolabeled probe and electrophoresis.
Oligonucleotides and Transfections.
The Stat3 antisense (5'-GCT CCA GCA TCT GCT GCT TC-3') or control mismatch oligonucleotides (5'-GCT CCA ATA CCC GTT GCT TC-3') were synthesized using phosphorothioate chemistry. To increase stability, oligonucleotides were synthesized with 2'-O-methoxyethyl modification of the five terminal nucleotides (underlined; Ref. 29
). Transfections were carried out by the Lipofectamine-Plus method as described by the supplier (Life Technologies, Inc.). Briefly, DU145 cells were seeded at 2 x 106 cells/10-cm tissue-culture plates 18 h before transfection in RPMI 1640 supplemented with 10% FBS. Immediately before transfection, cells were washed once with PBS. Cells were transfected for 3 h with Lipofectamine-Plus (LF+) alone, with LF+/Stat3 antisense oligonucleotides, LF+/Stat3 mismatch oligonucleotides, or not transfected. The final concentration of the oligonucleotides was 250 nM. The transfection was terminated by aspirating the transfection medium, washing the cells one time with PBS and adding fresh RPMI 1640 containing 10% FBS. Another 24 h later, the nonadherent cells were washed off and the remaining cells were lysed EMSA or Western blot analysis.
Apoptosis and Proliferation Assays.
For apoptosis assays, DU145 cells were treated for 24 h with Lipofectamine-Plus alone or with Stat3 antisense or Stat3 mismatch control oligonucleotides. Treated cells were stained with an antibody specific for activated caspase-3 using the supplier’s protocol (Becton Dickinson PharMingen) and analyzed by flow cytometry. For proliferation assays, cell numbers were determined by counting with a hemocytometer using trypan blue exclusion.
Immunohistochemical Detection of Phospho-Stat3.
Cytospins from LNCaP, DU145, and PC3 cells lines without treatment, LNCaP cells stimulated with IL-6 (10 ng/ml), and DU145 cells transfected with Lipofectamine-Plus (LF+) alone, with LF+/Stat3 antisense oligonucleotides, or LF+/Stat3 mismatch oligonucleotides were fixed in 95% ethanol for 10 min. Forty-five pairs of matched primary prostate tumors with normal tissue were fixed in 10% neutral-buffered formalin and embedded in serial 3–4-mm paraffin blocks. Five-µm thick sections were stained with H&E for histological examination. Representative histological sections and cytospins were then immunostained to localize Stat3. Immunostaining for phospho-Stat3 was performed using a rabbit antihuman polyclonal antibody (Phospho-Tyr705-Stat3; Cell Signaling, Beverly, MA). As negative controls, rabbit immunoglobulins (Vector, Burlingame, CA) were used to replace primary antibody. The 5-µm thick tissue sections described above were deparaffinized and hydrated in deionized water. The immunohistochemical staining was performed manually at room temperature, using the avidin-biotin-peroxidase complex method (Vectastatin Elite ABC kit; Vector Lab). Briefly, pretreatment for antigen retrieval with a pressure cooker involved heating cytospins and tissue sections with a microwave oven, in 250 ml of unmasking solution (Vector Lab) for 20 min at high-power level, followed by 20 min of cooling time. Slides were then treated with 0.025% trypsin in 50 mM Tris (pH 7.6) for 5 min at 37°C without prewarming. Endogenous peroxidase and nonspecific background staining were blocked by incubating slides with 3% hydrogen peroxide for 10 min. After washing with PBS for 5 min, slides were blocked with normal serum and 3% BSA for 10 min, followed by incubation with the phospho-Stat3 primary antibody, at a dilution of 1:400, overnight at 4°C. After rinsing with PBS for 5 min, slides were incubated with a biotinylated secondary antibody for 60 min and washed again. After washing with PBS for 5 min, slides were incubated with avidin-biotin complex for 1 h and washed again. Chromogen was developed with 3,3-diaminobenzidine (DAB Substrate kit for peroxidase; Vector Lab) or Nova-red (Nova-red Substrate kit for peroxidase; Vector Lab). All of the slides were lightly counterstained with hematoxylin for 30 s before dehydration and mounting. The pathologists (L. B. M., N. A.) evaluated immunohistochemical reactions in the cell lines and primary tumors.
Image Analysis Cytometry.
The computer-assisted CAS-200 Image Analysis System was used to quantify immunohistochemical staining of positive nuclei/total number of cells (x 100 for percent). A minimum of 300 nonoverlapping and well-preserved cells were measured within at least 10 adjacent x400 magnification fields in each sample. Quantification of nuclei positive for phospho-Stat3 staining was assessed in tumor and normal prostatic gland cells. The CAS-200 system operator was unaware of the Stat3 DNA-binding results by EMSA at the time of evaluation. Data were analyzed for statistical significance using the Wilcoxon signed-rank test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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revealed elevated Stat3 DNA-binding activity, to different extents, in 82% (37 of 45) of primary adenocarcinoma specimens examined (Lanes T1 through T45) relative to matched adjacent nontumor tissues (Lanes N1 through N45). It is also notable that 29% (13 of 45) of adjacent nontumor tissues exhibited Stat3 DNA-binding activity higher than basal levels present in a majority of nontumor prostate tissues. Only one case displayed no detectable Stat3 activity in the tumor tissue (Lane T5*). Thus, a high frequency of constitutive Stat3 activation is associated with primary prostate tumors in vivo, consistent with a role for activated Stat3 signaling in prostate cancer.
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, Stat3 DNA-binding activity is present in nuclear extracts prepared from all three of the prostate cancer cell lines examined. DU145 cells have the highest level, whereas LNCaP cells have the lowest level and are the least malignant, in agreement with the earlier findings of Ni et al. (30)
. Antibodies to Stat3, but not Stat1, could block and supershift this DNA-binding activity, confirming that the activity corresponds to Stat3 in all three cell lines (data not shown). We examined whether IL-6 could induce further Stat3 DNA-binding activity in the LNCaP cells. Treatment of serum-starved LNCaP cells with 10 ng/ml IL-6 induced a time-dependent activation of Stat3 (Fig. 2B)
. Antibodies to Stat3, but not Stat1, blocked this DNA-binding activity, confirming that it corresponds to Stat3. Consistent with the cell line data, antibody supershift analysis of all 45 patient tumor specimens from Fig. 1
demonstrated that the DNA-binding activity corresponds to Stat3 and not Stat1 (Fig. 2C
, and data not shown).
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, phospho-Stat3 is detected in the nucleus with different intensities in all three of the human prostate cancer cell lines examined. The intensity of red stain (Nova-Red) corresponds to the level of active phospho-Stat3, whereas the blue stain (hematoxylin) is the counterstain. The levels of phospho-Stat3 immunoreactivity correlate well with the levels of Stat3 DNA-binding activity as measured by EMSA in these cells (compare Figs. 2A
and 3A
), with DU145 cells having the highest and LNCaP cells having the lowest levels.
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below). After optimization of the immunohistochemical protocol, we used the phospho-Stat3 antibody to investigate the levels and cytological locations of activated Stat3 in the tumor specimens from prostate carcinoma patients. Immunohistochemistry demonstrated that low levels of nuclear phosphorylated Stat3 are restricted to the basal epithelial cells in normal (N) tissue adjacent to tumor tissue (Fig. 3B)
. In contrast, elevated levels of phospho-Stat3 are detected primarily in the nuclei of epithelial tumor cells in tumor (T) specimens (Fig. 3B)
. To quantify these observations, epithelial cells within the glands of prostate samples were analyzed by computer-assisted image analysis and the data are summarized in Fig. 3C
. Statistically, the difference in phospho-Stat3 levels between the malignant specimens and nontumor specimens was highly significant (P < 0.001) using the Wilcoxon signed-rank test. Increased Stat3 tyrosine phosphorylation in the tumor cells detected by immunohistochemistry correlates well with elevated Stat3-DNA binding activities as detected by EMSA for these specimens (Fig. 1
, and data not shown).
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, increased phospho-Stat3 levels were highly correlated (P < 0.007) with more malignant tumor specimens exhibiting Gleason scores 
7. By contrast, there was no significant correlation between levels of phospho-Stat3 and either initial serum PSA levels or clinical stage (Table 1)
. It was not possible to assess the correlation between levels of phospho-Stat3 and established parameters of disease progression (e.g., PSA levels after prostatectomy) because of the short time of follow-up after treatment of this cohort of patients. Thus, higher phospho-Stat3 levels correlate well with more aggressive disease in prostate cancer as determined by Gleason score at the time of radical prostatectomy.
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, shows greatly decreased Stat3 DNA-binding activity in the DU145 cells transfected with Stat3 antisense oligo (Lane 3) compared with nontransfected cells (Lane 1), Lipofectamine-Plus (LF+) transfection reagent alone (Lane 2), or Stat3 mismatch control oligonucleotide (Lane 4). Antibodies to Stat3 could block and supershift this DNA-binding activity, confirming that the activity corresponds to Stat3. Western blot analysis of total Stat3 protein levels (Fig. 4B)
demonstrates that Stat3 antisense (Lane 3) but not mismatch oligonucleotides (Lane 2) were able to substantially diminish Stat3 protein expression.
To establish that the immunohistochemical assay described above (Fig. 3)
is specific for phospho-Stat3, we blocked Stat3 expression using antisense Stat3 oligonucleotide. This block in Stat3 expression should ablate specifically the phospho-Stat3 antigen and, thus, represents a highly rigorous control for antibody specificity. Immunostaining of cytospins prepared from DU145 cells treated with Stat3 antisense oligonucleotide demonstrated that the phospho-Stat3 staining was abolished (Fig. 4C)
. In addition, stimulation of LNCaP cells with IL-6 resulted in increased phospho-Stat3 immunostaining, as expected (Fig. 4D)
. These results demonstrate that the antibody and immunohistochemical methods used are specific for phospho-Stat3 and that the immunostaining results correlate well with Stat3 DNA-binding activities as measured by EMSA analysis.
Blocking Stat3 Signaling Inhibits Growth and Survival of Prostate Cancer Cells.
Fig. 5A
shows that treatment of DU145 cells with LF+/Stat3 antisense oligonucleotide induced significant growth inhibition that correlated well with the inactivation of Stat3 as measured by EMSA. The control mismatch oligonucleotide had an intermediate effect on cell growth, consistent with the partial effect on Stat3 DNA-binding activity (compare Figs. 4A
and 5A
). This intermediate effect of the mismatch oligonucleotide may reflect nonspecific toxicity. To determine whether down-modulation of Stat3 by using antisense oligonucleotide could induce apoptosis in prostate tumor cells, we transfected DU145 cells and, 12 h later, measured apoptosis using an early marker for apoptosis, caspase 3 activation (Fig. 5B)
. Results demonstrate a 3-fold increase in apoptosis of the cells treated with Stat3 antisense oligonucleotide compared with the Lipofectamine-Plus (LF+) reagent alone or Stat3 mismatch control oligonucleotide. Collectively, these findings indicate that activated Stat3 signaling is required for the growth and survival of prostate cancer cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence suggest that IL-6 that are produced by tumor cells themselves or by adjacent stromal cells can modulate the growth of prostate tumor cells in an autocrine or paracrine manner (16 , 32, 33, 34) . The expression of IL-6 and its receptor has been detected not only in human prostate cancer cell lines but, more significantly, in human prostate carcinoma and benign prostate hyperplasia (BPH) specimens obtained directly from patients (35 , 36) . In addition, levels of IL-6 are elevated in the serum of patients with hormone-refractory and metastatic prostate carcinoma (35 , 36) . These findings suggest that the elevated Stat3 activation levels that we observed in prostate tumor tissues may arise from autocrine and/or paracrine stimulation by IL-6. Furthermore, it is possible that the elevated Stat3 activation detected in some of the adjacent nontumor tissues could also be induced by IL-6. Consistent with this possibility, levels of phospho-Stat3 were generally higher in nontumor tissues that were closer to the regions of tumor than in those that were farther away, suggesting that factors such as IL-6 secreted by tumor cells may induce Stat3 activation in adjacent tissues.5
Our findings are consistent with recent studies showing that IL-6 stimulates prostate cancer cell growth and activation of Stat3 signaling (37) and that the inhibition of Stat3 signaling blocks the growth of prostate cancer cells (30) . In contrast, other studies (38 , 39) suggested that Stat3 signaling correlated with IL-6-induced growth arrest and differentiation of prostate cancer cells. The apparent discrepancy between the latter (38 , 39) and the former studies (30 , 37) as well as the present study may be explained by other factors, such as androgens, which could modulate responses to Stat3 signaling. In this context, it may be significant that IL-6 undergoes a transition from being growth inhibitory to being growth stimulatory during progression to hormone-refractory prostate cancer (40) . Moreover, recent studies indicate that IL-6 induces androgen receptor-mediated gene regulation through the Stat3 protein (41) , raising the possibility that IL-6-induced Stat3 activation may contribute to the development of hormone-refractory prostate cancer. Consistent with a growth-stimulatory role of Stat3 in prostate cancer (30 , 37 , 41) , our results demonstrate that the direct inhibition of Stat3 signaling by antisense Stat3 oligonucleotides blocks the growth and survival of these cells.
Our findings reported here are also in agreement with recent studies showing that phospho-Stat3 levels are elevated in malignant prostate cells in vivo (42)
, further supporting a role for activated Stat3 in prostate cancer. To our knowledge, this is the first study in which the levels of activated Stat3 have been evaluated in parallel by a biochemical assay for Stat3 DNA-binding activity and an immunohistochemical assay for phospho-Stat3 in human tumors. Furthermore, levels of Stat3 activation were evaluated in the context of clinicopathological characteristics in a group of 45 patients with primary prostate adenocarcinoma. Our analysis demonstrates that higher levels of Stat3 activation are associated with higher Gleason scores (7), which is indicative of more aggressive and poorly differentiated tumors. This observation may be valuable for identification and management of high-risk prostate cancer patients, especially those with high Gleason scores. Additional studies are necessary to evaluate the prognostic significance of Stat3 activation in prostate cancer patients and correlation with other prognostic markers, such as Ki-67, angiogenesis and DNA ploidy, as well as clinical outcome by follow-up serum PSA levels (1, 2, 3, 4, 5)
.
Earlier studies showed that activated Stat3 signaling contributes to the growth and survival of diverse human cancer cells (24) such as multiple myeloma (15) , breast carcinoma (43) , melanoma (44) , and head and neck squamous carcinoma (45) . Thus, the development of therapeutic agents that target Stat3 signaling represents a potentially new treatment approach for prostate cancer and other human tumors (46) . With the determination of phospho-Stat3 levels by immunohistochemistry, it might be possible to identify and stratify patients who may respond to therapies specifically targeting Stat3 signaling. This immunohistochemical assay has certain advantages over EMSA including cost effectiveness, reproducibility, and adaptability to the clinical setting. Importantly, the specificity of this immunohistochemical assay was confirmed by specifically ablating Stat3 expression with the use of antisense Stat3 oligonucleotides. Another major advantage is that the specific cell types (i.e., tumor and nontumor cells) that have activated Stat3 can be distinguished by immunohistochemical but not by molecular approaches such as EMSA or Western blot analysis. Moreover, inexpensive and rapid follow-up of clinical outcome and response to treatment can be performed based on repeated immunohistochemical assays using limited amounts of tissue material including core biopsies. It is important to note, however, that rapid processing (less than 15 min from surgical removal) is essential for preserving the in vivo phosphorylation state of Stat3 protein and, thus, for obtaining reliable data on Stat3 activation in tumor specimens. The immunohistochemical protocol is also critical to expose the epitope without denaturing it too much so that it can be recognized by the antibodies. In conclusion, these results encourage further exploration of the potential prognostic and therapeutic value of elevated Stat3 activation in patients with prostate carcinoma and other tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Cancer Institute Grants CA55652 and CA82533 (to R. J.) and by the Dr. Mildred Scheel Stiftung fuer Krebsforschung (to R. B.). 
2 L. B. M. and R. B. contributed equally to this work.
3 To whom requests for reprints should be addressed, at Molecular Oncology Program, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612. Phone: (813) 979-6725; Fax: (813) 632-1436; E-mail: richjove{at}moffitt.usf.edu
4 The abbreviations used are: STAT, signal transducer(s) and activator(s) of transcription; IL, interleukin; FBS, fetal bovine serum; EMSA, electrophoretic mobility shift assay; hSIE, high affinity variant of the sis-inducible element.
5 L. B. Mora, unpublished observations.
Received 10/21/01. Accepted 9/18/02.
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I. Bellezza, H. Neuwirt, C. Nemes, I. T. Cavarretta, M. Puhr, H. Steiner, A. Minelli, G. Bartsch, F. Offner, A. Hobisch, et al. Suppressor of Cytokine Signaling-3 Antagonizes cAMP Effects on Proliferation and Apoptosis and Is Expressed in Human Prostate Cancer Am. J. Pathol., December 1, 2006; 169(6): 2199 - 2208. [Abstract] [Full Text] [PDF]
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W.-C. Li, S.-L. Ye, R.-X. Sun, Y.-K. Liu, Z.-Y. Tang, Y. Kim, J. G. Karras, and H. Zhang Inhibition of Growth and Metastasis of Human Hepatocellular Carcinoma by Antisense Oligonucleotide Targeting Signal Transducer and Activator of Transcription 3 Clin. Cancer Res., December 1, 2006; 12(23): 7140 - 7148. [Abstract] [Full Text] [PDF]
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Z. Duan, J. E. Bradner, E. Greenberg, R. Levine, R. Foster, J. Mahoney, and M. V. Seiden SD-1029 Inhibits Signal Transducer and Activator of Transcription 3 Nuclear Translocation. Clin. Cancer Res., November 15, 2006; 12(22): 6844 - 6852. [Abstract] [Full Text] [PDF]
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K. W. Kim, R. W. Mutter, C. Cao, J. M. Albert, E. T. Shinohara, K. R. Sekhar, and B. Lu Inhibition of signal transducer and activator of transcription 3 activity results in down-regulation of Survivin following irradiation. Mol. Cancer Ther., November 1, 2006; 5(11): 2659 - 2665. [Abstract] [Full Text] [PDF]
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 Z. S. Nagy, H. Rui, S. M. Stepkowski, J. Karras, and R. A. Kirken A Preferential Role for STAT5, not Constitutively Active STAT3, in Promoting Survival of a Human Lymphoid Tumor J. Immunol., October 15, 2006; 177(8): 5032 - 5040. [Abstract] [Full Text] [PDF]
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 R. P Singh and R. Agarwal Mechanisms of action of novel agents for prostate cancer chemoprevention. Endocr. Relat. Cancer, September 1, 2006; 13(3): 751 - 778. [Abstract] [Full Text] [PDF]
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A. Kotha, M. Sekharam, L. Cilenti, K. Siddiquee, A. Khaled, A. S. Zervos, B. Carter, J. Turkson, and R. Jove Resveratrol inhibits Src and Stat3 signaling and induces the apoptosis of malignant cells containing activated Stat3 protein. Mol. Cancer Ther., March 1, 2006; 5(3): 621 - 629. [Abstract] [Full Text] [PDF]
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H. Shao, X. Xu, N. Jing, and D. J. Tweardy Unique structural determinants for stat3 recruitment and activation by the granulocyte colony-stimulating factor receptor at phosphotyrosine ligands 704 and 744. J. Immunol., March 1, 2006; 176(5): 2933 - 2941. [Abstract] [Full Text] [PDF]
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N. Jing, Q. Zhu, P. Yuan, Y. Li, L. Mao, and D. J. Tweardy Targeting signal transducer and activator of transcription 3 with G-quartet oligonucleotides: a potential novel therapy for head and neck cancer. Mol. Cancer Ther., February 1, 2006; 5(2): 279 - 286. [Abstract] [Full Text] [PDF]
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T. Gritsko, A. Williams, J. Turkson, S. Kaneko, T. Bowman, M. Huang, S. Nam, I. Eweis, N. Diaz, D. Sullivan, et al. Persistent Activation of Stat3 Signaling Induces Survivin Gene Expression and Confers Resistance to Apoptosis in Human Breast Cancer Cells Clin. Cancer Res., January 1, 2006; 12(1): 11 - 19. [Abstract] [Full Text] [PDF]
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E. B. Haura, Z. Zheng, L. Song, A. Cantor, and G. Bepler Activated Epidermal Growth Factor Receptor-Stat-3 Signaling Promotes Tumor Survival In vivo in Non-Small Cell Lung Cancer Clin. Cancer Res., December 1, 2005; 11(23): 8288 - 8294. [Abstract] [Full Text] [PDF]
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S. M. Pulukuri, C. S. Gondi, S. S. Lakka, A. Jutla, N. Estes, M. Gujrati, and J. S. Rao RNA Interference-directed Knockdown of Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor Inhibits Prostate Cancer Cell Invasion, Survival, and Tumorigenicity in Vivo J. Biol. Chem., October 28, 2005; 280(43): 36529 - 36540. [Abstract] [Full Text] [PDF]
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Q. Lin, R. Lai, L. R. Chirieac, C. Li, V. A. Thomazy, I. Grammatikakis, G. Z. Rassidakis, W. Zhang, Y. Fujio, K. Kunisada, et al. Constitutive Activation of JAK3/STAT3 in Colon Carcinoma Tumors and Cell Lines: Inhibition of JAK3/STAT3 Signaling Induces Apoptosis and Cell Cycle Arrest of Colon Carcinoma Cells Am. J. Pathol., October 1, 2005; 167(4): 969 - 980. [Abstract] [Full Text] [PDF]
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J. Turkson, S. Zhang, L. B. Mora, A. Burns, S. Sebti, and R. Jove A Novel Platinum Compound Inhibits Constitutive Stat3 Signaling and Induces Cell Cycle Arrest and Apoptosis of Malignant Cells J. Biol. Chem., September 23, 2005; 280(38): 32979 - 32988. [Abstract] [Full Text] [PDF]
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L. Gao, L. Zhang, J. Hu, F. Li, Y. Shao, D. Zhao, D. V. Kalvakolanu, D. J. Kopecko, X. Zhao, and D.-Q. Xu Down-Regulation of Signal Transducer and Activator of Transcription 3 Expression Using Vector-Based Small Interfering RNAs Suppresses Growth of Human Prostate Tumor In vivo Clin. Cancer Res., September 1, 2005; 11(17): 6333 - 6341. [Abstract] [Full Text] [PDF]
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 Z. Zheng, G. Bepler, A. Cantor, and E. B. Haura Small Tumor Size and Limited Smoking History Predicts Activated Epidermal Growth Factor Receptor in Early-Stage Non-small Cell Lung Cancer Chest, July 1, 2005; 128(1): 308 - 316. [Abstract] [Full Text] [PDF]
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K. Schlessinger and D. E. Levy Malignant Transformation but not Normal Cell Growth Depends on Signal Transducer and Activator of Transcription 3 Cancer Res., July 1, 2005; 65(13): 5828 - 5834. [Abstract] [Full Text] [PDF]
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J. V. Alvarez, P. G. Febbo, S. Ramaswamy, M. Loda, A. Richardson, and D. A. Frank Identification of a Genetic Signature of Activated Signal Transducer and Activator of Transcription 3 in Human Tumors Cancer Res., June 15, 2005; 65(12): 5054 - 5062. [Abstract] [Full Text] [PDF]
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Z Culig, H Steiner, G Bartsch, and A Hobisch Mechanisms of endocrine therapy-responsive and -unresponsive prostate tumours Endocr. Relat. Cancer, June 1, 2005; 12(2): 229 - 244. [Abstract] [Full Text] [PDF]
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H. Song, R. Wang, S. Wang, and J. Lin A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells PNAS, March 29, 2005; 102(13): 4700 - 4705. [Abstract] [Full Text] [PDF]
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 M. F. McCarty Targeting Multiple Signaling Pathways as a Strategy for Managing Prostate Cancer: Multifocal Signal Modulation Therapy Integr Cancer Ther, December 1, 2004; 3(4): 349 - 380. [Abstract] [PDF]
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M. Thomas, C. E. Finnegan, K. M.-A. Rogers, J. W. Purcell, A. Trimble, P. G. Johnston, and M. P. Boland STAT1: A Modulator of Chemotherapy-induced Apoptosis Cancer Res., November 15, 2004; 64(22): 8357 - 8364. [Abstract] [Full Text] [PDF]
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V. J. Assikis, K.-A. Do, S. Wen, X. Wang, J. H. Cho-Vega, S. Brisbay, R. Lopez, C. J. Logothetis, P. Troncoso, C. N. Papandreou, et al. Clinical and Biomarker Correlates of Androgen-Independent, Locally Aggressive Prostate Cancer with Limited Metastatic Potential Clin. Cancer Res., October 15, 2004; 10(20): 6770 - 6778. [Abstract] [Full Text] [PDF]
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B. E. Barton, T. F. Murphy, P. Shu, H. F. Huang, M. Meyenhofen, and A. Barton Novel single-stranded oligonucleotides that inhibit signal transducer and activator of transcription 3 induce apoptosis in vitro and in vivo in prostate cancer cell lines Mol. Cancer Ther., October 1, 2004; 3(10): 1183 - 1191. [Abstract] [Full Text] [PDF]
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N. Jing, Y. Li, W. Xiong, W. Sha, L. Jing, and D. J. Tweardy G-Quartet Oligonucleotides: A New Class of Signal Transducer and Activator of Transcription 3 Inhibitors That Suppresses Growth of Prostate and Breast Tumors through Induction of Apoptosis Cancer Res., September 15, 2004; 64(18): 6603 - 6609. [Abstract] [Full Text] [PDF]
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 H. Siavash, N.G. Nikitakis, and J.J. Sauk SIGNAL TRANSDUCERS AND ACTIVATORS OF TRANSCRIPTION: INSIGHTS INTO THE MOLECULAR BASIS OF ORAL CANCER Critical Reviews in Oral Biology & Medicine, September 1, 2004; 15(5): 298 - 307. [Abstract] [Full Text] [PDF]
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R. D. Read, E. A. Bach, and R. L. Cagan Drosophila C-Terminal Src Kinase Negatively Regulates Organ Growth and Cell Proliferation through Inhibition of the Src, Jun N-Terminal Kinase, and STAT Pathways Mol. Cell. Biol., August 1, 2004; 24(15): 6676 - 6689. [Abstract] [Full Text] [PDF]
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H. Li, T. J. Ahonen, K. Alanen, J. Xie, M. J. LeBaron, T. G. Pretlow, E. L. Ealley, Y. Zhang, M. Nurmi, B. Singh, et al. Activation of Signal Transducer and Activator of Transcription 5 in Human Prostate Cancer Is Associated with High Histological Grade Cancer Res., July 15, 2004; 64(14): 4774 - 4782. [Abstract] [Full Text] [PDF]
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A. Moroz, C. Eppolito, Q. Li, J. Tao, C. H. Clegg, and P. A. Shrikant IL-21 Enhances and Sustains CD8+ T Cell Responses to Achieve Durable Tumor Immunity: Comparative Evaluation of IL-2, IL-15, and IL-21 J. Immunol., July 15, 2004; 173(2): 900 - 909. [Abstract] [Full Text] [PDF]
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D. L. Silver, H. Naora, J. Liu, W. Cheng, and D. J. Montell Activated Signal Transducer and Activator of Transcription (STAT) 3: Localization in Focal Adhesions and Function in Ovarian Cancer Cell Motility Cancer Res., May 15, 2004; 64(10): 3550 - 3558. [Abstract] [Full Text] [PDF]
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 J. Kim, R. M. Adam, K. R. Solomon, and M. R. Freeman Involvement of Cholesterol-Rich Lipid Rafts in Interleukin-6-Induced Neuroendocrine Differentiation of LNCaP Prostate Cancer Cells Endocrinology, February 1, 2004; 145(2): 613 - 619. [Abstract] [Full Text] [PDF]
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B. E. Barton, J. G. Karras, T. F. Murphy, A. Barton, and H. F-S. Huang Signal transducer and activator of transcription 3 (STAT3) activation in prostate cancer: Direct STAT3 inhibition induces apoptosis in prostate cancer lines Mol. Cancer Ther., January 1, 2004; 3(1): 11 - 20. [Abstract] [Full Text]
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B. He, L. You, K. Uematsu, K. Zang, Z. Xu, A. Y. Lee, J. F. Costello, F. McCormick, and D. M. Jablons SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer PNAS, November 25, 2003; 100(24): 14133 - 14138. [Abstract] [Full Text] [PDF]
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J. W. Mandell Phosphorylation State-Specific Antibodies: Applications in Investigative and Diagnostic Pathology Am. J. Pathol., November 1, 2003; 163(5): 1687 - 1698. [Abstract] [Full Text] [PDF]
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J. D. Khoury, L. J. Medeiros, G. Z. Rassidakis, M. A. Yared, P. Tsioli, V. Leventaki, A. Schmitt-Graeff, M. Herling, H. M. Amin, and R. Lai Differential Expression and Clinical Significance of Tyrosine-phosphorylated STAT3 in ALK+ and ALK- Anaplastic Large Cell Lymphoma Clin. Cancer Res., September 1, 2003; 9(10): 3692 - 3699. [Abstract] [Full Text] [PDF]
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