We study the IL-6/JAK/Stat3 pathway in a variety of tumor model systems. In particular we are interested in the cross-talk between tumor cells and the myriad of cells within the microenvironment which plays a critical role in the metastatic behavior of tumors.
The IL-6/Stat3 Signaling Pathway Promotes Mammary Tumorigenesis and Metastases
IL-6 and its signaling target, tyrosine phosphorylated Stat3, are heterogeneously expressed in primary breast tumors. The highest expression of these proteins are within tumor emboli (areas of ymphovascular invasion) and on the leading edge of tumors in association with stroma, endothelial cells and immune cells. Breast cancer derived cell lines which express high levels of IL-6 are dependent on this growth factor for migration, in vitro, in vivo growth and metastatic potential. IL-6 signals through Stat3 which we demonstrate to be required for both primary tumor growth and metastases. Finally, enhancing IL-6 levels in cancer cell lines markedly increases the metastatic potential of breast tumors.
Stat3 is Required for Mammary Tumorigenesis
In order to determine the functional requirement of Stat3 in mammary tumorigenesis, we have conditionally deleted Stat3 from the mammary gland in the context of two mouse breast cancer models (MMTV-PyMT and MMTV-Neu) whose invasive tumors express high pStat3. Furthermore, we will determine the consequences of blocking Stat3 activation in human derived breast cancer derived cell lines. Our preliminary data demonstrates that in vivo Stat3 deletion reduces the growth of MMTV-PyMT tumors concomitant with diminished angiogenesis and leukocyte recruitment. Tumor latency is not effected. However, survival was significantly increased in mice lacking Stat3. We have also shown that a lack of Stat3 leads to tumors which are very cystic. Unfortunately, our data was derived in mice which are notin a pure C57/B6 background (5 generations backcrossed) and therefore we cannot be confident of our data as there are significant differences in tumor latency and metastatic formation in different mouse strains. However, we have now backcrossed our mice (11 generations) to the C57/B6 background and are ready to confirm our preliminary observations in the mixed background. These findings demonstrate a requirement for Stat3 in both a murine model and xenograft models of mammary tumorigenesis.
Stat3 Mediates Expression of Autotaxin a Regulator of Migration in Breast Cancer
Signal transducer and activator of transcription 3 (Stat3) is constitutively activated in 37 percent of primary breast tumors and 63 percent 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 "leading edge" of tumors with relatively lower levels in the central portion of tumors. This observation led us to hypothesize that activated Stat3 through differential gene regulation may mediate metastatic spread of cancer cells. In order to identify Stat3 target genes involved in migration and metastasis we compared those genes that were differentially expressed in primary breast cancer samples as a function of pStat3 levels. We identified ENPP2 which encodes a secreted lysophospholipase which can mediate breast cancer cell migration, as a novel Stat3 target gene in breast cancer. A positive correlation between nuclear pStat3 and ATX was determined by IHC of primary breast cancer samples. Similarly, a positive correlation between high pStat3 levels and ATX expression was determined in several breast cancer derived cell lines. Inhibition of pStat3 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 is involved in the metastatic spread of breast cancer cells through the regulation of ATX.
Determine Whether Stat3C Expression in Mammary Epithelial Cells Promotes Chronic Inflammation and De Novo Tumorigenesis in the Mammary Gland
We have developed a novel mouse model that permits doxycycline-inducible Stat3C expression in the breast gland. We have demonstrated that enforced expression of Stat3C in the lactating mammary gland is sufficient to mediate involution. This result is consistent with the data demonstrating that Stat3 is required for involution during normal weaning (1). Interestingly, inducible expression of Stat3C did not mediate apopotis of the mammary gland which is observed 24-48 hours after pup removal in the normal mammary gland, but autophagy (another form of programmed cell death-survival). This is mediated in part through the upregulation of beclin-1 a required mediator of this process. This is the first demonstration that activated Stat3 can mediate the process of autophagy. Our original preliminary data which examined the consequences of Stat3C expression in the mammary gland revealed an increase in inflammatory cell recruitment to the mammary gland. Unfortunately, we have had difficulty repeating these observations as doxycycline inducible expression of Stat3C is not consistent from mouse to mouse. However, experiments performed for Aim2 have revealed that high doses of doxycycline (2mg/ml) in the food or water would lead to very transient and high levels of Stat3C but the protein would quickly turn over likely causing cell death. In contrast, 1000 fold lower doses led to sustained and high levels of expression. This mouse model has led to the discovery that activated Stat3 is sufficient to mediate mammary gland involution through the activation of autophagy in part by regulating beclin-1 protein levels. We hypothesize that Stat3 mediated autophagy may play a role in tumorigenesis. It has been demonstrated that patients with breast tumors expressing high levels of activated Stat3 are relatively resistant to neoadjuvant chemotherapy (2). Autophagy, a form of programmed cell death is activated during times of stress (starvation, toxic insults, transformation) which acts in part to prevent or delay cells from undergoing necrotic or apoptotic death by suppressing a number of metabolic pathways. Ultimately, if the stress is not relieved than a cell will indeed die. However, it has been suggested that autophagy may protect cancer cells from undergoing apoptosis in response to chemotherapy or radiation therapy(3). We are hypothesizing that Stat3 may regulate autophagy in cancer cells thereby conferring resistance to chemotherapy. Expression of high levels of activated Stat3 leads to rapid turn-over of the protein and lack of sustained expression in mammary epithelial cells. However, given our ability to titrate levels of expression, we hypothesize that lower levels will be expressed and recapitulate a pro-inflammatory phenotype in the mammary gland. We will investigate the mechanism of Stat3 mediated autophagy in not only the involuting mammary but also in cancer cells. We propose determining whether in cancer cells which express high levels of activated Stat3 whether chemotherapy or radiation induced cell death is in part affected by modulation of autophagy. By using knock-down strategies (well established in our laboratory) or inhibitors of IL-6/Jak/Stat3 signaling, we will determine whether Stat3 plays a role in the process. Regarding in vivo expression of activated Stat3 in the mammary gland we propose treating MMTV-rtTAxTetO-Stat3C double transgenic mice with varying (low) levels of doxycycline and determine the optimal concentration which gives rise to sustained expression of Stat3C.
Determine Whether Stat3C Expression Cooperates with Mammary Oncogenes in the Development and Progression of Mammary Tumorigenesis and Etastatic Spread
Human tumors are phenotypically heterogenous with regards to clinical behavior as a consequence of the step-wise acquisition of specific aberrantly regulated signaling pathways. We will therefore examine and determine the consequences of adding activated Stat3 to established murine mammary tumor models (MMTV-Myc and MMTV-PyMT). We will determine the cooperative effects of Stat3C on tumor latency, leukocyte recruitment, and histologic and metastatic progression.
We have established models whereby Stat3C expression can be induced in the mammary gland in the context of 2 mouse models: MMTV-Myc and MMTV-PyMT. Using conventional doses of doxycycline (2mg/ml in the mouse chow) we observed transient and high levels of Stat3C expression as determined by IHC and western blot. However, over time (several weeks) Stat3C expression was decreased and frequently lost and it appeared that the protein was turned over as some cells had residual staining for Stat3C (Flag-staining as this protein has a flag epitope tag on its COO- end) in a speckled pattern. Thus, no correlation could be made regarding tumorigenesis (latency, proliferation, metastatic potential) as a function of Stat3 expression. In order to address this problem we lowered the doxycycline concentration and determined that by administering 1000x less doxycycline (1mg/ml) we observe fairly uniform and high expression of Stat3C throughout the tumor. We have also isolated numerous cell lines from MMTV-PyMTxMMTV-rtTAxTetO-Stat3C mice and have demonstrate that these cells form tumors which can metastasize when injected into the mammary fat pad of syngeneic (FVB) mice. Furthermore, administration of low dose doxycycline leads to robust and sustained expression of Stat3C in vivo. We show in vitro that addition of doxycycline leads to an increase in the ability of these cells to invade and migrate in a Boyden Chamber assay and grow in suspension (suggestive of enhancing stem cell characteristics). Finally, we observe that primary tumor growth is enhanced 3-fold when Stat3C is expressed. These preliminary results demonstrate that inducible expression of activated Stat3 in the context of a tumor can enhance the tumors proliferative potential and in vitro migratory and invasive potential. We propose determining the mechanisms by which activated Stat3 can modulate tumorigenesis including epithelial specific effects as well as the ability of Stat3+ tumor cells to interact with stromal cells and immune cells to enhance tumorigenesis. We will specifically focus on recruitment of mesenchymal stem cells, dendritic cells, hematopoietic progenitor cells to the tumor site as well as to metastatic sites.
Differential IL-6/Stat3 Signaling as a Function of Cellular Context Mediates Ras-Induced Transformation
Tyrosine phosphorylated signal transducer and activator of transcription 3 (pStat3) is present in numerous cancers and is required for mediating tumorigenesis. Recently, non-tyrosine phosphorylated Stat3 was shown to have a transcriptional activating function and to mediate cell migration. The Ras oncogene mediates cellular transformation without evidence of pStat3 in cultured cells. Here the role of Stat3 in Ras transformed mammary epithelial cells (MCF10A-Ras) was examined by down-regulating Stat3 expression. Cellular migration, invasion, anchorage independent growth and tumorigenesis were largely abrogated in the Stat3-reduced cells compared to control cells. Analysis of the MCF10A-Ras tumors revealed high levels of pStat3 and interleukin-6 (IL-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. Reducing IL-6 levels in the MCF10A-Ras cells abrogated tumorigenesis. By isolating Ras-expressing primary tumors and serially passaging these cells in two-dimensional culture resulted in a decrease in IL-6 and pStat3 levels. Thus, as a function of the context in which the Ras oncogene is expressed, can lead to IL-6/pStat3 signaling and a dependency on this transcription factor for migration, invasion and tumorigenesis.
IL-6 Signaling Between Mesenchymal Stem Cells and Breast Cancer Cells
It has been shown that mesenchymal cells can enhance or promote the metastatic behavior of breast cancer cells. We are studying the role of IL-6/Stat3 signaling in this process and our preliminary data suggests that IL-6 mediated regulation of CXCR4 and SDF1 is responsible for these effects.
The Role of Constitutively Activated Stat3 in Stem Cells
We have developed a novel mouse model that permits doxycycline-inducible Stat3C expression throughout most cell types within the mouse. Strikingly inducible expression of Stat3C leads to an expansion of hematopoietic, skin and gastric stem cells. This phenotype is principally mediated through upregulation of IL-6.
IL-6/Jak2 Inhibition for the Treatment of Lung Cancer
Lung cancer is the leading cause of death from cancer worldwide. While the majority of lung cancers are due to tobacco abuse, a subset (~10 percent) of lung cancers occur in never-smokers and typically harbor a mutant form of the epidermal growth factor receptor (EGFR). Although most of these patients initially respond to targeted inhibitors of the EGFR enzyme they eventually develop resistance to the EGFR kinase inhibitors (and succumb to their disease) which in about 50 percent of cases is due to a second mutation within the EGFR. We recently determined that the Signal transducer and activator of transcription 3 (Stat3) protein is aberrantly activated in both the EGFR kinase inhibitor sensitive and resistant lung cancers harboring mutant forms of the EGFR as a consequence of enhanced production of the tumor promoting factor interluekin-6. The Stat3 molecule has been shown to play a critical tumor promoting role in a number of cancers by increasing the growth and blood supply to tumors as well as promoting resistance to chemotherapy. Importantly, we have recently characterized a novel inhibitor of the IL-6/Stat3 pathway which effectively blocks Stat3 activity and the growth of cultured lung cancer cells including those that are resistant to EGFR kinase inhibitors. We will determine whether this inhibitor (Jak inhibitor) will block the growth of lung cancers in mice injected with human cancer cells and in mouse models of lung cancer expressing the mutant forms of the EGFR (including those that are resistant to EGFR kinase inhibitors). In addition, we will test whether "removal" of IL-6 by genetic deletion can decrease or delay cancer development in mouse lung cancer models. The overall aim of this proposal is to develop the necessary pre-clinical evidence of targeting the IL-6/Jak/Stat3 pathway in lung cancers including those who have developed resistance to EGFR kinase inhibitors and chemotherapy. We believe that blocking this pathway is a potentially important and novel approach to treating patients with lung cancer.
