The Role of IL-6/Stat3 Signaling in Cancer Stem Cell Behavior: Cross-Talk between Cancer Cells and the Microenvironment
For example, we have determined that the Stat3 protein and its activating partner, IL-6 are expressed at very high levels on the invasive edge of a tumor. We hypothesize that these cells on the leading edge of a tumor are enriched in cancer stem cells which are capable of metastasizing to distant sites and are resistant to chemotherapy.
IL-6/Stat3 Signaling in Mammary Tumorigenesis
We observe that tumor cells within the central portion of a cancer are rounded and lack activated Stat3. In contrast cancer cells on the tumor edge have a spindle shape and express high levels of activated Stat3. The process of going from a rounded cell to a spindle like cell is called an epithelial to mesenchymal transition (EMT). It is hypothesized that these cells are enriched for cancer stem cells. Tumor cells within areas of vascular invasion are also spindle shaped and express high levels of activated Stat3. In addition, the cells comprising the microenvironment (stromal cells, blood vessel cells and immune cells) are all positive for activated Stat3. We have demonstrated that a specific subset of cells within the tumor microenvironment (called mesenchymal cells) promote the growth of adjacent cancer cells in particular cancer stem cells. We hypothesize that mesenchymal cells are recruited to a tumor which signals to the cancer cell in an IL-6/Stat3 dependent manner leading to enhanced growth, invasion and metastases through Stat3-dependent regulation of key genes involved in homing, migration, invasion, new blood vessel growth and cancer stem cell expansion. To test this hypothesis, we will examine the effects of inhibiting the IL-6/Stat3 pathway in both tumor cells and mesenchymal cells using model systems of primary tumorigenesis and metastases including homing of mesenchymal cells to tumors, induction of an epithelial to mesenchymal transition, enhanced tumorigenesis and metastases. We will also examine in non-metastatic mammary tumor models whether co-injections of tumor cells and mesenchymal stem cells can mediate metastatic progression. By blocking IL-6 signaling (or reducing levels of IL-6 and or Stat3) in both breast cancer derived cell lines and mesenchymal cells we will determine the significance of this pathway to these processes. We will demonstrate the functional consequences of the interleukin-6 signaling pathway in the “crosstalk” between tumor cells and the cells surrounding the tumor which significantly influences the behavior of a tumor (e.g. converting the tumor from an indolent one to a tumor which is highly metastatic and resistant to chemotherapy). The results of this work will establish the rationale for targeted therapies aimed at the IL-6 pathway in breast cancer.
The IL-6/Jak/Stat3 Pathway: Targeting Metastatic Breast Cancer
Breast cancer is the most common malignancy diagnosed among women worldwide. Clinically, breast cancer is characterized by the size of the tumor, the ability of the cells to spread to lymphnodes and expression of the estrogen and Her2neu receptors which dictates treatment options. However, many patients have cancers which cannot be treated with targeted anti-estrogen or anti-Her2neu therapies. Despite significant improvements in the diagnosis and treatment of this disease, tumor dormancy followed by distant recurrences accounts for 90 percent of all cancer deaths. This suggests that malignant cells have already disseminated at the time of diagnosis. Micrometastases in the blood and bone marrow are the principal targets for adjuvant chemotherapy and hormonal therapy. However, these metastatic cells frequently evade therapeutic interventions and eventually recur.
Of recent interest is the discovery that within a tumor, only a tiny subset of cancer cells have the potential to metastasize and grow in different locations (lung, bone, liver, etc.). These cells have been called “cancer stem cells” and do not express the typical proteins of “mature” breast cancer cells such as the estrogen, progesterone and Her2neu receptors and are therefore also called “triple negative” (estrogen, progesterone, Her2neu receptor negative) cancer cells. Importantly, these cells are relatively resistant to chemotherapy. Clearly understanding the molecular mechanisms underlying the development of metastatic disease is required in order to treat this potentially fatal disorder effectively.
During periods of stress (both physical and emotional) a protein called interleukin-6 (IL-6) is produced by many different cell types in an attempt to promote healing. In addition, IL-6 is also produced to high levels by certain breast cancer cells, in particular the “triple negative” breast cancer stem cells and those cells which have the capacity to metastasize. The IL-6 protein mediates its effects on cell behavior by first binding to the IL-6 receptor which is found on the surface of cells. Once the IL-6 receptor is engaged it activates an enzyme called Jak2 which subsequently modifies or turns-on a protein called Stat3. In normal cells Stat3 is turned on and off like a light switch in response to specific cues such as transient pulses of IL-6. In contrast to normal cells, we have determined that Stat3 is constitutively in the “on” or “activated” mode in ~50 percent of breast cancers. Furthermore, by genetically changing a normal Stat3 into one that is constitutively active, we can make a normal breast cell behave as a cancerous one. We have recently demonstrated that breast cancer cells harboring an activated Stat3 are addicted or dependent upon this protein for both growth and metastatic spread. Importantly, we have determined that abnormal signaling from the growth factor interleukin-6 (IL-6) and the enzyme Jak2 is the principal mechanism by which Stat3 is “turned-on” in breast cancers. Furthermore, we have shown that activated Stat3 can itself mediate the production of IL-6 which not only leads to persistent Stat3 activation but also recruits and activates inflammatory cells which stimulate tumor progression and metastatic spread.
One of the questions we are asking is whether IL-6 is capable of converting a dormant breast cancer cell into an actively growing one which can ultimately harm patients. In order to address this question we analyzed a number of human breast cancer derived cells which do not have the capacity to metastasize (e.g. defined by an inability to proliferate in the lung). We introduced the gene encoding IL-6 into one of these cell lines and observed that it now had acquired the capacity to metastasize and proliferate in the lung. Thus, by simply increasing levels of IL-6 in a cancer cell can mediate its transformation from a “dormant” cancer to one capable of rapidly proliferating in the lung. We are pursuing this line of research further by determining how much IL-6 is needed and for how long does a cancer cell need to “see” IL-6 in order to undergo this transformation. We will also determine whether IL-6 can mediate the transition from dormancy to growth in other sites such as bone and liver (two other very common sites of metastatic spread).
These findings led us to ask the important question of whether blocking IL-6 signaling in breast cancer cells could reverse or stop the growth of established metastases? In order to block IL-6 signaling we have taken multiple approaches. The first includes antibodies which recognize either the IL-6 cell surface receptor preventing IL-6 protein from binding or an antibody which recognizes IL-6 directly and effectively sequesters it away from the cell. The second approach targets the Jak2 enzyme, the critical enzyme which transmits the signal from the IL-6/IL-6R to the inside of the cell. Our preliminary data using both IL-6 receptor blocking antibodies and inhibitors of the Jak2 enzyme, strongly suggests that blocking this pathway abrogates growth of tumors and inhibits their metastatic spread. We propose confirming these preliminary but exciting findings and determining whether they can inhibit the growth or further spread of established metastatic disease. Clearly, these latter studies are very relevant to the treatment of patients with existing metastatic disease. In addition, we propose combining these novel inhibitors with standard chemotherapeutic agents used for the treatment of metastatic breast cancer in the hopes of completely eliminating metastatic disease.
A common clinical scenario in the treatment of patients with metastatic breast cancer is an initial “response” to a particular drug with the eventual outgrowth of cancer cells which are “resistant” to the drug. It is clear that chronic exposure of a tumor to a drug has not led to curative regimens perhaps based on our lack of understanding on how to dose the drug optimally. Using mathematical methods, new ways of dosing chemotherapies were established which were shown to markedly increase the efficacy of standard chemotherapeutic agents without increasing toxicity. This was achieved by giving the chemotherapy at a greater dose rate: administering the drug at shorter intervals which reduced the regrowth of tumors between cycles of therapy and prevented the cancer cells from acquiring ways of by-passing or resisting the effects of chemotherapy. By applying these recently described methods by Norton et al. whereby chemotherapy dosing schedules can be optimized by using mathematical models of cancer growth, we propose determining the optimal dosing schedule for both the Jak2 inhibitor and the antibodies against the IL-6 receptor.
We will establish the anti-tumor effects of blocking the IL-6 receptor or inhibiting the Jak2 enzyme and determine the mechanisms by which this is occurring. Furthermore, we will determine the role of blocking the IL-6 pathway on prevention of metastases but more importantly on the treatment of existing metastases. Finally we will identify the optimal manner for administering these compounds which will be of tremendous clinical value and will set the stage for novel approaches to designing clinical trials.
Stat3 Target Genes: A Regulator of Metastases
Signal transducer and activator of transcription 3 (Stat3) is constitutively activated in 37 percent of primary breast tumors and 6 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.
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.
Activated Stat3 Regulates 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.