Persistently activated or tyrosine-phosphorylated STAT3 (pSTAT3) is found in 50% of lung adenocarcinomas. pSTAT3 is found in primary adenocarcinomas and cell lines harboring somatic-activating mutations in the tyrosine kinase domain of EGFR. Treatment of cell lines with either an EGFR inhibitor or an src kinase inhibitor had no effect on pSTAT3 levels, whereas a pan-JAK inhibitor (P6) blocked activation of STAT3 and inhibited tumorigenesis. Cell lines expressing these persistently activated mutant EGFRs also produced high IL-6 levels, and blockade of the IL-6/gp130/JAK pathway led to a decrease in pSTAT3 levels. In addition, reduction of IL-6 levels by RNA interference led to a decrease in tumorigenesis. Introduction of persistently activated EGFR into immortalized breast epithelial cells led to tumorigenesis, IL-6 expression, and STAT3 activation, all of which could be inhibited with P6 or gp130 blockade. Furthermore, inhibition of EGFR activity in multiple cell lines partially blocked transcription of IL-6 and concurrently decreased production and release of IL-6. Finally, immunohistochemical analysis revealed a positive correlation between pSTAT3 and IL-6 positivity in primary lung adenocarcinomas. Therefore, mutant EGFR could activate the gp130/JAK/STAT3 pathway by means of IL-6 upregulation in primary human lung adenocarcinomas, making this pathway a potential target for cancer treatment.

Persistently activated Stat3 is found in many different cancers, including Ϸ60% of breast tumors. Here, we demonstrate that a constitutively activated Stat3 transforms immortalized human mammary epithelial cells and that this oncogenic event requires the activity of matrix metalloproteinase-9 (MMP-9). By immunohistochemical analysis, we observe a positive correlation between strong MMP-9 expression and tyrosine phosphorylated Stat3 in primary breast cancer specimens. These results demonstrate a relationship between activated Stat3 and MMP-9 in breast oncogenesis.

Introduction Signal transducer and activator of transcription 3 (Stat3) is constitutively tyrosine-phosphorylated in approximately 50% of primary breast carcinomas. A number of different mechanisms responsible for Stat3 activation, including abnormal activation of receptor tyrosine kinases, Src, and Janus kinases (Jaks), have been implicated in breast cancer.

Signal transducers and activators of transcription 3 (STAT3) is a transcription factor that is aberrantly activated in many cancer cells. Constitutively activated STAT3 is oncogenic, presumably as a consequence of the genes that it differentially regulates. Activated STAT3 correlated with elevated cyclin D1 protein in primary breast tumors and breast cancer-derived cell lines. Cyclin D1 mRNA levels were increased in primary rat-, mouse-, and human-derived cell lines expressing either the oncogenic variant of STAT3 (STAT3-C) or vSrc, which constitutively phosphorylates STAT3. Mutagenesis of STAT3 binding sites within the cyclin D1 promoter and chromatin immunoprecipitation studies showed an association between STAT3 and the transcriptional regulation of the human cyclin D1 gene.

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IntroductionTyrosine phosphorylated signal transducer and activator of transcription 3 (pStat3) is expressed in numerous cancers and is required for mediating tumorigenesis. Autocrine and paracrine interleukin (IL)-6 signaling is the principal mechanism by which Stat3 is persistently phosphorylated in epithelial tumors including breast, lung, colon and gastric cancer. The Ras oncogene mediates cellular transformation without evidence of pStat3 in cultured cells. However, non-tyrosine phosphorylated Stat3 was shown to function as a transcriptional activator, localize to the mitochondria and regulate ATP synthesis and mediate cell migration. Here we examined the role of Stat3 in Ras mediated transformation.MethodsHa-rasV12 transformed mammary epithelial cells (MCF10A-Ras) cells were transduced with a Stat3shRNA, IL-6shRNA and/or treated with inhibitors of Janus kinases (JAKs) to examine the role of the IL-6 signaling pathway in Ras mediated migration, invasion and tumorigenesis.ResultsCellular migration, invasion, anchorage independent growth and tumorigenesis were largely abrogated in the Stat3-reduced cells compared to control cells. Analysis of MCF10A-Ras tumors revealed high levels of pStat3 and interleukin-6. Tumors derived from transgenic MMTV-K-Ras mice were also found to express pStat3 and IL-6. MCF10A-Ras cells, when grown in a three-dimensional Matrigel culture system revealed the appearance of the junctional protein E-Cadherin as a consequence of reducing Stat3 levels or inhibiting Stat3 activity. Decreasing IL-6 levels in the MCF10A-Ras cells abrogated tumorigenesis and reduced cell migration. By isolating Ras-expressing primary tumors and serially passaging these cells in two-dimensional culture led to a decrease in IL-6 and pStat3 levels with the reappearance of E-Cadherin.ConclusionsThe cellular and environmental context can lead to differential IL-6/pStat3 signaling and a dependency on this cytokine and transcription factor for migration, invasion and tumorigenesis.

We determined that signal transducer and activator of transcription 3 (Stat3) is tyrosine phosphorylated in 37% of primary breast tumors and 63% of paired metastatic axillary lymph nodes. Examination of the distribution of tyrosine phosphorylated (pStat3) in primary tumors revealed heterogenous expression within the tumor with the highest levels found in cells on the edge of tumors with relatively lower levels in the central portion of tumors. In order to determine Stat3 target genes that may be involved in migration and metastasis, we identified those genes that were differentially expressed in primary breast cancer samples as a function of pStat3 levels. In addition to known Stat3 transcriptional targets (Twist, Snail, Tenascin-C and IL-8), we identified ENPP2 as a novel Stat3 regulated gene, which encodes autotaxin (ATX), a secreted lysophospholipase which mediates mammary tumorigenesis and cancer cell migration. A positive correlation between nuclear pStat3 and ATX was determined by immunohistochemical analysis of primary breast cancer samples and matched axillary lymph nodes and in several breast cancer derived cell lines. Inhibition of pStat3 or reducing Stat3 expression led to a decrease in ATX levels and cell migration. An association between Stat3 and the ATX promoter, which contains a number of putative Stat3 binding sites, was determined by chromatin immunoprecipitation. These observations suggest that activated Stat3 may regulate the migration of breast cancer cells through the regulation of ATX.

is that L~(~-B U O )~A I H~, Li(t-BuO),AIH, and LiA1H4 are in slow equilibrium according to eq 2. Confirmation of this suggestion was sought by approaching the equilibrium from the opposite direction. If this equilibrium is present, one should be able to start with a solution of Li(t-BuO),AIH and add LiA1H4 to reestablish the equilibrium. Spectrum G in Figures 2-4 was obtained in just such a manner. To a solution of Li(t-BuO),AIH (spectrum E in Figures 2, 4, and 5) was added a solution of LiA1H4 so that the new molar ratio was 1.7. Note that in the 27Al spectrum ( Figure 2) the broad singlet -21.9 ppm from LiAIH4 is gone and that in the I3C spectrum ( Figure 4) the singlet for Li(t-BuO),AIH is nearly gone while the singlet for L~(~-B U O )~A I H~ has reappeared.From the ratio of the integrated areas of the methyl singlets in the I3C spectrum one can obtain the ratio Rc:The 27Al spectra of solutions containing tert-butyl alcohol/LiA1H4 molar ratios between 0.5 and 2.0 consist of a broad singlet due to L~(~-B U O )~A I H~ and Li(t-BuO),AIH with a quintet from LiAIH4 imposed upon it. Using the instruments computer, one may subtract the LiAIH4 quintet from the spectra to obtain just the broad singlet whose area is proportional to the concentration of Li(t-BuO),A1H2 plus Li(t-BuO),AIH; the resulting broad singlets are shown in Figure 3. In this way one may determine the ratio RAI: [LiAIH,] [ L~(~-B U O )~A I H~] + [ Li(t-BuO),AIH] = RAl (4) Together, these ratios allow the calculation of the equilibrium constant [Li( t-BuO),AIH] 2[LiA1H4] [Li( ~-B U O )~A I H~] K , = (5) K , = ( 3 / 2 R~)~R~1 ( 3 / 2 & -I-1) Combining eq 3-5 one can obtain ( 6 )The data in Table I indicate an equilibrium constant of 2.2 X lo-'. Con c 1 us i o n sWe find that for molar ratios for tert-butyl alcohol/LiAIH, up to 2 the major tert-butoxyaluminate in solution is Li(t-B U O )~A I H~ and that it is in a slow equilibrium with LiA1H4 and Li(t-BuO),AIH. If Li(t-BuO)AIH, is present, its concentration must be low compared to the Li(t-BuO)2A1H2 concentration.Clearly the tert-butoxyaluminate system is not as simple a system as some have thought it to be.Acknowledgments. The support of the Department of Chemistry at The University of Toledo is gratefully acknowledged. We thank Professors J. Fry and J. Gano for helpful discussion.Abstract: In this article the equilibrium constant and enthalpy for formation of the dioxygen adduct of the bis(sa1icyliden-iminato-3-propyl)methylaminocobalt(II) complex (CoSMDPT) are reported in methylene chloride solution (-9.8 kcal mol-'). When small amounts of 2,2,2-trifluoroethanoI (TFE) are added, extra stabilization of the dioxygen adduct is found. The measured enthalpy for adduct formation increases to -1 1.8 kcal mol-'. Infrared frequency shift studies of the 0-H stretching vibration indicate that TFE undergoes a hydrogen-bonding interaction with the coordinated dioxygen. An enthalpy-frequency shift, AuOH, relation provides an estimate of a 6.6 kcal mol-' hydrogen bond strength. This is one of the few instances i...