Puja Kachroo

IL-27 inhibits epithelial-mesenchymal transition and angiogenic factor production in a STAT1-dominant pathway in human non-small cell lung cancer

BackgroundInterleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. Epithelial–mesenchymal transition (EMT) and angiogenesis are key processes in carcinogenesis. Although IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. In this study, we investigated the role of IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human non-small cell lung carcinoma (NSCLC) cells.MethodsSTAT activation following IL-27 exposure was measured in human NSCLC cell lines. Expression of epithelial (E-cadherin, γ-catenin) and mesenchymal (N-cadherin, vimentin) markers were assessed by Western blot analysis. Production of pro-angiogenic factors (VEGF, IL-8/CXCL8, CXCL5) were examined by ELISA. Cell motility was examined by an in vitro scratch and transwell migration assays. Selective inhibitors of STAT1 (STAT1 siRNAs) and STAT3 (Stattic) were used to determine whether both STAT1 and STAT3 are required for IL-27 mediated inhibition of EMT and secretion of angiogenic factors.ResultsOur results demonstrate that IL-27 stimulation in NSCLC resulted in 1) STAT1 and STAT3 activation in a JAK-dependent manner, 2) development of epithelial phenotypes, including a decrease in the expression of a transcriptional repressor for E-cadherin (SNAIL), and mesenchymal marker (vimentin) with a reciprocal increase in the expression of epithelial markers, 3) inhibition of cell migration, and 4) reduced production of pro-angiogenic factors. STAT1 inhibition in IL-27–treated cells reversed the IL-27 effect with resultant increased expression of Snail, vimentin and the pro-angiogenic factors. The inhibition of STAT3 activation had no effect on the development of the epithelial phenotype.ConclusionIL-27 induces mesenchymal to epithelial transition and inhibits the production of pro-angiogenic factors in a STAT1–dominant pathway. These findings highlight the importance of STAT1 in repressing lung carcinogenesis and describe a new anti-tumor mechanism of IL-27.

Single-Institution, Multidisciplinary Experience with Surgical Resection of Primary Chest Wall Sarcomas

Introduction: Primary chest wall sarcomas are rare mesenchymal tumors and their mainstay of therapy is wide surgical resection. We report our single-institution, multidisciplinary experience with full-thickness resection for primary chest wall sarcomas. Methods: A retrospective review of our prospectively maintained databases revealed that 51 patients were referred for primary chest wall sarcomas from 1990 to 2009. Results: All patients required resections that included rib and/or sternum. Twenty-nine patients (57%) had extended resections beyond the chest wall. Forty-two patients (82%) required prosthetic reconstruction and 17 patients (33%) had muscle flap coverage. Overall, 51% (26/51) of patients received neoadjuvant therapy. Seventy-three percent (11/15) of high-grade soft tissue sarcomas, 77% (10/13) of high-risk bony sarcomas, and 67% (4/6) of desmoid tumors were treated with induction therapy. Negative margins were obtained in 46 patients (90%). There were no perioperative mortalities. Eight patients (16%) experienced complications. Local recurrence and metastasis was detected in 14 and 23%. Five-year overall and disease-free survivals were 66% and 47%, respectively. Favorable prognostic variables for survival included age ⩽50 years, tumor volume ⩽200 cm3, desmoid tumor, bony tumor, chondrosarcoma, and low-grade soft tissue sarcoma. Conclusions: We report our multidisciplinary experience with primary chest wall sarcomas that included induction therapy in the majority of high-risk soft tissue and bony sarcomas and desmoid tumors. Despite aggressive preoperative treatments, acceptable surgical results with low morbidity and mortality can be achieved. Neoadjuvant systemic therapy may reduce local and distant recurrence and improve overall survival.

Ossicular motion related to middle ear transmission delay in gerbil.

The middle ear transmits sound efficiently from the air in the ear canal (EC) to the fluid filled cochlea. In gerbil, middle ear transmission produces a constant pressure gain between the EC and the cochlea of ∼25 dB from 2 to 40 kHz, and a delay-like phase corresponding to a ∼25-30 μs delay. The mechanisms by which the air-born signal is collected and delivered to the cochlea are not thoroughly understood, and the source of the delay is controversial. We investigated these issues by observing ossicular motion along a single line of sight, roughly parallel to the EC and perpendicular to the stapes footplate. Measurements were made at the umbo, the long process of the manubrium, across the malleus-incus joint, at the long process of the incus, and the stapes head. While the overall delay between EC pressure and stapes velocity was fairly constant with frequency, subcomponents of the delay were frequency dependent. Up to ∼17 kHz, most of the overall delay was between the EC and umbo with a much smaller contribution along the ossicles, whereas in the range from ∼17 to 30 kHz, more of the overall delay was along the ossicles.

Combination Treatment with Apricoxib and IL-27 Enhances Inhibition of Epithelial-Mesenchymal Transition in Human Lung Cancer Cells through a STAT1 Dominant Pathway

BACKGROUND The cyclooxygenase 2 (COX-2) pathway has been implicated in the molecular pathogenesis of many malignancies, including lung cancer. Apricoxib, a selective COX-2 inhibitor, has been described to inhibit epithelial-mesenchymal transition (EMT) in human malignancies. The mechanism by which apricoxib may alter the tumor microenvironment by affecting EMT through other important signaling pathways is poorly defined. IL-27 has been shown to have anti-tumor activity and our recent study showed that IL-27 inhibited EMT through a STAT1 dominant pathway. OBJECTIVE The purpose of this study is to investigate the role of apricoxib combined with IL-27 in inhibiting lung carcinogenesis by modulation of EMT through STAT signaling. METHODS AND RESULTS Western blot analysis revealed that IL-27 stimulation of human non-small cell lung cancer (NSCLC) cell lines results in STAT1 and STAT3 activation, decreased Snail protein and mesenchymal markers (N-cadherin and vimentin) and a concomitant increase in expression of epithelial markers (E-cadherin, β-and γ-catenins), and inhibition of cell migration. The combination of apricoxib and IL-27 resulted in augmentation of STAT1 activation. However, IL-27 mediated STAT3 activation was decreased by the addition of apricoxib. STAT1 siRNA was used to determine the involvement of STAT1 pathway in the enhanced inhibition of EMT and cell migration by the combined IL-27 and apricoxib treatment. Pretreatment of cells with STAT1 siRNA inhibited the effect of combined IL-27 and apricoxib in the activation of STAT1 and STAT3. In addition, the augmented expression of epithelial markers, decreased expression mesenchymal markers, and inhibited cell migration by the combination treatment were also inhibited by STAT1 siRNA, suggesting that the STAT1 pathway is important in the enhanced effect from the combination treatment. CONCLUSION Combined apricoxib and IL-27 has an enhanced effect in inhibition of epithelial-mesenchymal transition and cell migration in human lung cancer cells through a STAT1 dominant pathway.

GITR agonist enhances vaccination responses in lung cancer

An immune tolerant tumor microenvironment promotes immune evasion of lung cancer. Agents that antagonize immune tolerance will thus aid the fight against this devastating disease. Members of the tumor necrosis factor receptor (TNFR) family modulate the magnitude, duration and phenotype of immune responsiveness to antigens. Among these, GITR expressed on immune cells functions as a key regulator in inflammatory and immune responses. Here, we evaluate the GITR agonistic antibody (DTA-1) as a mono-therapy and in combination with therapeutic vaccination in murine lung cancer models. We found that DTA-1 treatment of tumor-bearing mice increased: (i) the frequency and activation of intratumoral natural killer (NK) cells and T lymphocytes, (ii) the antigen presenting cell (APC) activity in the tumor, and (iii) systemic T-cell specific tumor cell cytolysis. DTA-1 treatment enhanced tumor cell apoptosis as quantified by cleaved caspase-3 staining in the tumors. DTA-1 treatment increased expression of IFNγ, TNFα and IL-12 but reduced IL-10 levels in tumors. Furthermore, increased anti-angiogenic chemokines corresponding with decreased pro-angiogenic chemokine levels correlated with reduced expression of the endothelial cell marker Meca 32 in the tumors of DTA-1 treated mice. In accordance, there was reduced tumor growth (8-fold by weight) in the DTA-1 treatment group. NK cell depletion markedly inhibited the antitumor response elicited by DTA-1. DTA-1 combined with therapeutic vaccination caused tumor rejection in 38% of mice and a 20-fold reduction in tumor burden in the remaining mice relative to control. Mice that rejected tumors following therapy developed immunological memory against subsequent re-challenge. Our data demonstrates GITR agonist antibody activated NK cell and T lymphocyte activity, and enhanced therapeutic vaccination responses against lung cancer.

Chest Wall Sarcomas are Accurately Diagnosed by Image-Guided Core Needle Biopsy

Objective: Sarcomas are rare mesenchymal malignancies. Accurate preoperative diagnosis is a prerequisite in considering investigational or institutional management algorithms that include neoadjuvant treatment. We reviewed our experience using core needle biopsy for chest wall sarcomas. Methods: A retrospective review of our sarcoma databases revealed that 40 core needle biopsies and 35 tumor resections were performed in 34 patients, with chest wall musculoskeletal tumors, referred to the University of California, Los Angeles from 1991 to 2010. Primary, metastatic, or recurrent sarcomas involving the sternum, ribs, and soft tissues of the chest wall were evaluated for (1) adequacy of tissue from image-guided core needle biopsies and (2) accuracy in determining malignancy, histological subtype, and sarcoma grade. Results: Twenty-eight of the 40 needle biopsy samples (70%) were adequate for histopathological analysis. Forty-two percent of nondiagnostic findings occurred due to insufficient tissue, whereas the remainder had sufficient tissue, but the pathologist was unable to determine specific histology. Excluding the nondiagnostic samples, the accuracy in determining malignancy, histological subtype, and grade in sarcomas was 100, 92, and 87%, respectively. The sensitivity and specificity of determining malignancy and high-grade sarcomas were 100, 100, 77, and 100%, respectively. There were no complications from the image-guided biopsies. Conclusions: We demonstrated that image-guided core needle biopsy when performed and reviewed by experienced radiologists and musculoskeletal pathologists is a safe and accurate diagnostic technique for chest wall sarcomas. Core needle biopsy should be considered in the multidisciplinary approach to chest wall musculoskeletal tumors, especially when induction therapy is considered.

Abstract 339: Apricoxib, a selective COX-2 inhibitor, suppresses IL-27-mediated STAT3 activation and potentiates its inhibition of epithelial to mesenchymal transition in human non-small cell lung cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction The cyclooxygenase 2 (COX-2) pathway has been implicated in the molecular pathogenesis of many cancers. Apricoxib, a selective COX-2 inhibitor, has recently been demonstrated to inhibit epithelial to mesenchymal transition (EMT) in human malignancies. EMT is a critical process in cancer progression and metastasis whereby epithelial cells undergo changes to a migratory, mesenchymal phenotype. The mechanism by which apricoxib may alter the tumor microenvironment by impacting other important pathways in EMT is poorly defined. To investigate this concept, we utilized Interleukin-27 (IL-27), a member of the IL-12 family, which signals through the JAK-STAT pathway and activates transcriptional factors with opposing roles in carcinogenesis, STAT1 (tumor suppressor) and STAT3 (oncogene). IL-27 has been shown to have anti-tumor activity, but understanding of its mechanism is limited. We previously demonstrated that IL-27 activates both STAT1 and STAT3 pathways in human non-small cell lung cancer (NSCLC) and that the balance in STAT1 and STAT3 activation is important in inhibiting EMT. Here, we studied the effect of apricoxib on IL-27-mediated STAT activation and EMT inhibition. We hypothesize that apricoxib modulates STAT1 and STAT3 activation and regulates EMT. Methods A human NSCLC cell line, A549, was pre-treated with apricoxib (80nM-10µM) for up to 24 hours prior to IL-27 stimulation (50ng/mL). Activation of STAT1 and STAT3 proteins, demonstrated by tyrosine phosphorylation, was measured by Western Blot analysis. Additionally, epithelial markers (E-cadherin, γ-catenin, β-catenin) and mesenchymal markers (N-cadherin, vimentin, snail) were evaluated also by Western blotting. Results IL-27 alone induced phosphorylation of STAT1 and STAT3 proteins compared to untreated control. Pre-treatment with apricoxib resulted in decreased levels of IL-27-mediated STAT1 and STAT3 phosphorylation compared to the IL-27 alone group. Apricoxib alone did not cause STAT1 or STAT3 activation. IL-27 treatment alone increased the levels of E-cadherin, γ-catenin, and β-catenin and decreased the levels of N-cadherin, vimentin, and snail compared to untreated control. Pre-treatment with apricoxib for 24 hours prior to IL-27 exposure resulted in potentiation of IL-27-mediated reduction of N-cadherin, vimentin, and snail and further increased the levels of E-cadherin when compared to the IL-27 alone group. There appeared to be no impact on the expression of γ-catenin or β-catenin with the addition of apricoxib to IL-27. Conclusions Apricoxib inhibits IL-27-mediated activation of STAT1 and STAT3, and potentiates the IL-27-mediated inhibition of epithelial to mesenchymal transition. These findings suggest that apricoxib may regulate EMT through modulation of the STAT pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 339. doi:1538-7445.AM2012-339

Abstract 3425: Interleukin-27 inhibits epithelial mesenchymal transition in lung carcinogenesis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Interleukin-27 (IL-27), the newest member of the IL-6/IL-12 family, is secreted by antigen presenting cells and has anti-tumor activity through its activation of the JAK-STAT pathway. Transcriptional factors STAT1 and STAT3 have been implicated in the balance of tumor suppressor and oncogenic functions. Epithelial mesenchymal transition (EMT) is a process whereby cells undergo changes from a highly polarized epithelial phenotype to a mesenchymal, migratory phenotype, which plays an important role in tumor metastasis. The role of IL-27 in EMT was evaluated in lung carcinogenesis. Human bronchial epithelial cells (HBECs), genetically modified HBEC to over-express Snail and K-ras (HBEC-Snail, HBEC-K-ras), and non-small cell lung cancer (NSCLC) lines (A549, H2122) were treated with IL-27 at multiple time points. Phosphorylation of STAT1 and STAT3 measured by Western Blot demonstrated activation of both pathways in these cell lines treated with IL-27. However, the STAT1 pathway was attenuated in HBEC-K-ras cells exposed to IL-27. Western analysis of markers for epithelial to mesenchymal transition (EMT) (E-cadherin, γ-catenin, N-cadherin, Vimentin, and Snail) showed up-regulation of E-cadherin and γ-catenin, important for the epithelial phenotype, which peaked 8 hours after IL-27 exposure, while markers important for the mesenchymal phenotype (N-cadherin, Vimentin, and Snail) were all down-regulated at similar time points. The addition of a STAT3 inhibitor to IL-27 treatment led to continued down-regulation of Vimentin beyond 8 hours. A wound healing functional assay to study cell migration after IL-27 treatment of cancer cell lines for up to 48 hours, demonstrated overall decrease in cell migration. In summary, IL-27 inhibits epithelial mesenchymal transition in NSCLCs and oncogenically manipulated HBECs through the regulation of STAT1 and STAT3 pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3425. doi:10.1158/1538-7445.AM2011-3425

Abstract 4268: Interleukin-27 inhibits angiogenesis in non-small cell lung cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Interleukin-27 (IL-27), the newest member of the IL-6/IL-12 family, is secreted by antigen presenting cells and has anti-tumor activity through its activation of the JAK-STAT pathway. Transcriptional factors STAT1 and STAT3 have been implicated in the balance of tumor suppressor and oncogenic functions. Angiogenesis is a critical process during carcinogenesis. We investigated the role of IL-27 in tumor angiogenesis in lung cancer. Human bronchial epithelial cells (HBECs), genetically modified HBEC to over express Snail and K-ras (HBEC-Snail, HBEC-K-ras), and human non-small cell lung cancer (NSCLC) lines (A549, H2122) were treated with IL-27 at multiple time points. Phosphorylation of STAT1 and STAT3 measured by Western Blot after IL-27 exposure demonstrated activation of both pathways in these cell lines. Protein production of an important pro-angiogenic factor, vascular endothelial growth factor (VEGF), was measured by ELISA and Western analysis. After exposure to IL-27, there was a 60-80% decrease in VEGF protein expression of VEGF by ELISA (confirmed by Western analysis in the NSCLC lines but not in the HBECs or their genetically modified derivatives). The IL-27 down regulation of VEGF was not increased by the addition of a STAT3 inhibitor. In summary, IL-27 inhibits VEGF production in NSCLC through a mechanism independent of STAT3, suggesting that STAT1 may play a dominant role in IL-27 mediated angiogenesis suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4268. doi:10.1158/1538-7445.AM2011-4268