Lanxi Song

Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis

Non-receptor and receptor tyrosine kinases, such as Src and EGF receptor (EGFR), are major inducers of vascular endothelial growth factor (VEGF), one of the most potent mediators of angiogenesis. While tyrosine kinases signal through multiple pathways, signal transducer and activation of transcription 3 (Stat3) is a point of convergence for many of these and is constitutively activated with high frequency in a wide range of cancer cells. Here, we show that VEGF expression correlates with Stat3 activity in diverse human cancer cell lines. An activated Stat3 mutant (Stat3C) up-regulates VEGF expression and stimulates tumor angiogenesis. Stat3C-induced VEGF up-regulation is abrogated when a Stat3-binding site in the VEGF promoter is mutated. Furthermore, interrupting Stat3 signaling with dominant-negative Stat3 protein or Stat3 antisense oligonucleotide in tumor cells down-regulates VEGF expression. Consistent with an important role of Stat3 in VEGF up-regulation induced by various oncogenic tyrosine kinases, v-Src-mediated VEGF expression is inhibited when Stat3 signaling is blocked. Moreover, chromatin immunoprecipitation assays indicate that Stat3 protein binds to the VEGF promoter in vivo and mutation of a Stat3-binding site in the VEGF promoter abrogates v-Src-induced VEGF promoter activity. These studies provide evidence that the VEGF gene is regulated directly by Stat3 protein, and indicate that Stat3 represents a common molecular target for blocking angiogenesis induced by multiple signaling pathways in human cancers.

Src kinases as therapeutic targets for cancer

Src family kinases (SFKs) have a critical role in cell adhesion, invasion, proliferation, survival, and angiogenesis during tumor development. SFKs comprise nine family members that share similar structure and function. Overexpression or high activation of SFKs occurs frequently in tumor tissues and they are central mediators in multiple signaling pathways that are important in oncogenesis. SFKs can interact with tyrosine kinase receptors, such as EGFR and the VEGF receptor. SFKs can affect cell proliferation via the Ras/ERK/MAPK pathway and can regulate gene expression via transcription factors such as STAT molecules. SFKs can also affect cell adhesion and migration via interaction with integrins, actins, GTPase-activating proteins, scaffold proteins, such as p130CAS and paxillin, and kinases such as focal adhesion kinases. Furthermore, SFKs can regulate angiogenesis via gene expression of angiogenic growth factors, such as fibroblast growth factor, VEGF, and interleukin 8. On the basis of these important findings, small-molecule SFK inhibitors have been developed and are undergoing early phase clinical testing. In preclinical studies these agents can suppress tumor growth and metastases. The agents seem to be safe in humans and could add to the therapeutic arsenal against subsets of cancers.

Stat3 regulates genes common to both wound healing and cancer

Wound healing and cancer are both characterized by cell proliferation, remodeling of extracellular matrix, cell invasion and migration, new blood vessel formation, and modulation of blood coagulation. The mechanisms that link wound healing and cancer are poorly understood. We report here that Stat3, a common signaling mechanism involved in oncogenesis and tissue injury, regulates a common set of genes involved in wound healing and cancer. Using oligonucleotide gene arrays and quantitative real-time PCR, we evaluated changes in global gene expression resulting from expression of Stat3 in lung epithelial cells. We report here previously uncharacterized genes induced by Stat3 implicated in signaling pathways common to both wound healing and cancer including cell invasion and migration, angiogenesis, modulation of coagulation, and repression of interferon-inducible genes. Consistent with these results, we found increased Stat3 activity associated with wound healing in chronically inflamed mouse lungs and increased Stat3 activity was identified at the leading edge of lung tumors invading adjacent nontumor stroma. These findings provide a molecular basis for understanding cancer as a deregulation of normal wound healing processes.

Activation of Stat3 by receptor tyrosine kinases and cytokines regulates survival in human non-small cell carcinoma cells

Overexpression of receptor tyrosine kinases including the epidermal growth factor receptor (EGF-R) as well as nonreceptor tyrosine kinases, such as Src, have been implicated in the formation of human lung cancers. In addition, cytokines like interleukin-6 (IL-6) have been demonstrated to modulate lung cancer cell growth and elevated levels of IL-6 have been shown to be an adverse prognostic factor for patients with lung cancer. Despite a large body of evidence pointing to their potential importance, few direct studies into the role of signal transducers and activators of transcription (STAT) pathways in human lung cancer have been undertaken. Here we demonstrate that multiple nonsmall cell lung cancer cell lines demonstrate constitutive Stat3 DNA-binding activity. Stat3 DNA-binding activity is specifically upregulated by the addition of epidermal growth factor (EGF), IL-6, and hepatocyte-derived growth factor (HGF). Furthermore, the stimulation of Stat3 DNA-binding activity by EGF requires the activity of EGF-R tyrosine kinase as well as Src-kinase, while the upregulation of Stat3 activity by IL-6 or HGF requires only Src-kinase activity. Treatment of A549 lung cancer cells with PD180970 or SU6656, both pharmacological inhibitors of Src-kinase, resulted in reduced Src and Stat3 activity, cell cycle arrest in G2, and reduced viability of cells accompanied by induction of apoptosis. Treatment of Stat3-positive A549 and H358 cells with antisense Stat3 oligonucleotides results in complete loss of Stat3 DNA-binding activity and apoptosis, while Stat3-positive H1299 cells remained healthy. Finally, an adenoviral vector expressing a dominant-negative Stat3 isoform results in loss of Stat3 DNA-binding activity, apoptosis, and reduced cellular viability. These results demonstrate a role of Stat3 in transducing survival signals downstream of tyrosine kinases such as Src, EGF-R, and c-Met, as well as cytokines such as IL-6, in human nonsmall cell lung cancers.

A chemical and phosphoproteomic characterization of dasatinib action in lung cancer

We describe a strategy to comprehend signaling pathways active in lung cancer cells and targeted by dasatinib employing chemical proteomics to identify direct interacting proteins combined with immunoaffinity purification of tyrosine phosphorylated peptides corresponding to activated tyrosine kinases. We identified nearly 40 different kinase targets of dasatinib. These include SFK members (LYN, SRC, FYN, LCK, YES), non-receptor tyrosine kinases (FRK, BRK, ACK), and receptor tyrosine kinases (Ephrin receptors, DDR1, EGFR). Using quantitative phosphoproteomics we identified peptides corresponding to autophosphorylation sites of these tyrosine kinases that are inhibited in a concentration-dependent manner by dasatinib. Using drug resistant gatekeeper mutants, we show that SFK kinases, particularly SRC and FYN, as well as EGFR are relevant targets for dasatinib action. The combined mass spectrometry based approach described here provides a system-level view of dasatinib action in cancer cells and suggests both functional targets and rationale combinatorial therapeutic strategies.

Activated Epidermal Growth Factor Receptor ^ Stat-3 Signaling Promotes Tumor Survival In vivo in Non ^ Small Cell Lung Cancer

Purpose: Signal transducers and activators of transcription 3 (Stat3), a member of the STAT family of transcription factors, regulates multiple oncogenic pathways, including pathways regulating tumor cell survival. We evaluated Stat3 activation in early stage non–small cell lung cancers (NSCLC) and how this relates to upstream epidermal growth factor receptor (EGFR) activation, tumor apoptosis, and prognosis. Experimental Design: High-density tissue microarrays using tissues from 176 surgically resected NSCLC were evaluated for expression of phosphorylated Stat3 (pStat3) and epidermal growth factor receptor (pEGFR) along with tumor apoptosis. Using NSCLC cell lines, we evaluated how pStat3 expression relates to EGFR mutations and sensitivity of cells to gefitinib. Results: We identified nuclear pStat3 expression in 54% of tumors. pStat3 expression was correlated with smaller tumors (P < 0.0001) and with limited smoking history (P = 0.02). We identified a trend toward higher pStat3 expression in adenocarcinomas compared with other tumor histology (P = 0.09). No relationship was found between pStat3 and prognosis following surgical resection. Importantly, we found a strong positive correlation between pEGFR expression and pStat3 expression (P <0.0001) and an inverse correlation between pStat3 and apoptosis (P = 0.01) consistent with less apoptosis in tumors expressing high amounts of pStat3. Cell lines with mutant EGFR have increased levels of pStat3 compared with cell lines without mutant EGFR and this correlates with their sensitivity to gefitinib. Finally, antisense-mediated knockdown of Stat3 induces apoptosis in EGFR mutant lung cancer cells. Conclusions: Early-stage NSCLC tumors have activated EGFR-Stat3 signaling with low apoptosis. Our findings suggest that pStat3 expression may be helpful in identifying patients appropriate for treatment with EGFR tyrosine kinase inhibitors.

Dasatinib (BMS-354825) selectively induces apoptosis in lung cancer cells dependent on epidermal growth factor receptor signaling for survival.

Mutations of the epidermal growth factor receptor (EGFR) selectively activate Akt and signal transducer and activator of transcription (STAT) pathways that are important in lung cancer cell survival. Src family kinases can cooperate with receptor tyrosine kinases to signal through downstream molecules, such as phosphatidylinositol 3-kinase/PTEN/Akt and STATs. Based on the importance of EGFR signaling in lung cancer, the known cooperation between EGFR and Src proteins, and evidence of elevated Src activity in human lung cancers, we evaluated the effectiveness of a novel orally bioavailable Src inhibitor dasatinib (BMS-324825) in lung cancer cell lines with defined EGFR status. Here, we show that cell fate (death versus growth arrest) in lung cancer cells exposed to dasatinib is dependent on EGFR status. In cells with EGFR mutation that are dependent on EGFR for survival, dasatinib reduces cell viability through the induction of apoptosis while having minimal apoptotic effect on cell lines with wild-type (WT) EGFR. The induction of apoptosis in these EGFR-mutant cell lines corresponds to down-regulation of activated Akt and STAT3 survival proteins. In cell lines with WT or resistant EGFR mutation that are not sensitive to EGFR inhibition, dasatinib induces a G(1) cell cycle arrest with associated changes in cyclin D and p27 proteins, inhibits activated FAK, and prevents tumor cell invasion. Our results show that dasatinib could be effective therapy for patients with lung cancers through disruption of cell growth, survival, and tumor invasion. Our results suggest EGFR status is important in deciding cell fate in response to dasatinib.

Mcl-1 regulates survival and sensitivity to diverse apoptotic stimuli in human non-small cell lung cancer cells.

Overexpression of anti-apoptotic Bcl-2 family members and deregulation of the pathways that regulate pro-apoptotic family members have been observed in non-small cell lung cancers (NSCLC). Previous reports have identified both Bcl-2 and Bcl-xL proteins as survival factors in lung cancer cells since reductions in these proteins can induce apoptosis and sensitize lung cancer cells to apoptosis induced by chemotherapy agents. Myeloid cell leukemia-1 (Mcl-1), another member of the Bcl-2 family, has been found to be a critical survival factor in hematopoietic cells, yet little data exists for a role of Mcl-1 in human lung cancers. We used NSCLC cell lines to explore how Mcl-1 levels affect lung cancer cell survival and studied tumors from patients to determine expression patterns of Mcl-1. NSCLC cells express abundant Mcl-1 protein and depletion of Mcl-1 levels by antisense Mcl-1 oligonucleotides induces apoptosis in A549 and H1299 lung cancer cells. Reduction in Mcl-1 levels can sensitize lung cancer cells to apoptosis induced by cytotoxic agents as well as by ionizing radiation. Lung cancer cells overexpressing Mcl-1 are less sensitive to apoptosis induced by chemotherapeutic agents, ZD1839 (an inhibitor of EGFR tyrosine kinase) and Bcl-2 or Bcl-xL antisense oligonucleotides. We find that epidermal growth factor (EGF) can enhance Mcl-1 protein levels in an ERK-dependent manner. Signal transduction agents that reduce Mcl-1 levels correlated with their individual ability to induce apoptosis in lung cancer cells. Finally, NSCLC tumors taken directly from patients have elevated levels of Mcl-1 protein compared with normal adjacent lung tissue. Therefore, agents that target Mcl-1 can induce apoptosis and sensitize cells to apoptosis induced by cytotoxic agents. Mcl-1 protein is overexpressed in a subset of human NSCLC and enhanced levels of Mcl-1 may protect lung cancer cells from death induced by a variety of pro-apoptotic stimuli.

Phase I/II Study of the Src Inhibitor Dasatinib in Combination With Erlotinib in Advanced Non–Small-Cell Lung Cancer

PURPOSE Src family kinase (SFK) proteins are frequently activated in cancer and can coordinate tumor cell growth, survival, invasion, and angiogenesis. Given the importance of SFK signaling in cancer, known cooperation between SFK and epidermal growth factor receptor (EGFR) signaling, and efficacy of EGFR inhibitors, we performed a phase I trial combining dasatinib, an SFK and multikinase inhibitor, with erlotinib, an EGFR inhibitor, in patients with advanced non-small-cell lung cancer. PATIENTS AND METHODS Patients received erlotinib for 1 week before addition of dasatinib; pharmacokinetics were performed after weeks 1 and 2. Four cohorts were examined, including twice-daily and daily dasatinib dosing. Responses were assessed after 8 weeks. Plasma levels of angiogenic markers (vascular endothelial growth factor [VEGF], interleukin-8, and basic fibroblast growth factor [bFGF]) were determined before and during treatment. RESULTS Thirty-four patients were enrolled. The average duration of treatment was 73 days. The main adverse events include GI (diarrhea, anorexia, and nausea), skin rash, cytopenias, pleural effusions, and fatigue. No effect of escalating doses of dasatinib was observed on erlotinib pharmacokinetics. Two partial responses and one bone response were observed, and the disease control rate was 63%. Reductions in plasma VEGF and bFGF were observed, and reductions in VEGF correlated with disease control. CONCLUSION The combination of erlotinib and dasatinib is tolerable, with adverse effects consistent with the two agents. Disease control and inhibition of plasma angiogenesis markers were observed. Personalized strategies for deployment of SFK should receive further attention.

Direct repression of the Mcl-1 promoter by E2F1

E2F1 induces apoptosis via both p53-dependent and p53-independent mechanisms. The direct targets in the p53-independent pathway remain enigmatic; however, the induction of this pathway does not require the transactivation domain of E2F1. Using cells that are defective in p53 activation, we show that E2F1 potently represses the expression of Mcl-1 – an anti-apoptotic Bcl-2 family member whose depletion results in apoptosis. We also show that this transcriptional repression is direct and dependent upon E2F1s DNA-binding domain, but does not require the transactivation domain of E2F1. Consistent with this DNA binding requirement of E2F1 to repress Mcl-1, we show that E2F1 binds to the Mcl-1 promoter both in vitro and in vivo, and have identified the DNA element (−143/−117) within this promoter that is required for E2F1 binding and repression. Additionally, cell lines constitutively expressing Mcl-1 are resistant to E2F1-mediated apoptosis – suggesting that Mcl-1 downregulation is a necessary event in the p53-independent apoptotic process. Thus, we identify a p53 family-independent mechanism of E2F1-induced apoptosis in which E2F1 directly represses Mcl-1 expression.