Silencing the Snail-Dependent RNA Splice Regulator ESRP1 Drives Malignant Transformation of Human Pulmonary Epithelial Cells.

Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated, including in non-small cell lung cancer (NSCLC). Here, we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo Snail-mediated transformation relied upon silencing of the tumor-suppressive RNA splicing regulatory protein ESRP1. In clinical specimens of NSCLC, ESRP1 loss was documented in Snail-expressing premalignant pulmonary lesions. Mechanistic investigations showed that Snail drives malignant progression in an ALDH+CD44+CD24- pulmonary stem cell subset in which ESRP1 and stemness-repressing microRNAs are inhibited. Collectively, our results show how ESRP1 loss is a critical event in lung carcinogenesis, and they identify new candidate directions for targeted therapy of NSCLC.Significance: This study defines a Snail-ESRP1 cancer axis that is crucial for human lung carcinogenesis, with implications for new intervention strategies and translational opportunities. Cancer Res; 78(8); 1986-99. ©2018 AACR.

IL-7 inhibits fibroblast TGF-beta production and signaling in pulmonary fibrosis.

Based on studies by our group and others, we hypothesized that IL-7 may possess antifibrotic activities in an IFN-gamma-dependent and independent manner. Here, we have evaluated the antifibrotic therapeutic potential of IL-7 in both in vitro and in vivo pulmonary fibrosis models. IL-7 inhibited both TGF-beta production and signaling in fibroblasts and required an intact JAK1/STAT1 signal transduction pathway. IL-7-mediated inhibition of TGF-beta signaling was found to be associated with an increase in Smad7, a major inhibitory regulator in the SMAD family. In the presence of IL-7, Smad7 dominant negative fibroblasts restored TGF-beta-induced collagen synthesis, indicating that an IL-7-mediated increase in Smad7 suppressed TGF-beta signaling. Consistent with these in vitro findings, recombinant IL-7 decreased bleomycin-induced pulmonary fibrosis in vivo, independent of IFN-gamma. The antifibrotic activities of IL-7 merit further basic and clinical investigation for the treatment of pulmonary fibrosis.

Nonradioactive iodide effectively induces apoptosis in genetically modified lung cancer cells.

We assessed a nonradioactive approach to induce apoptosis in non-small cell lung cancer by a novel iodide uptake and retention mechanism. To enhance tumor apoptosis, we transduced non-small cell lung cancer cells with retroviral vectors containing the sodium iodide symporter (NIS) and thyroperoxidase (TPO) genes. Expression of NIS and TPO facilitated concentration of iodide in tumors. As a consequence of the marked increase in intracellular levels of iodide, apoptosis was seen in >95% of NIS/TPO-modified lung cancer cells. Intraperitoneal injection of potassium iodide resulted in significant tumor volume reduction in NIS/TPO-modified tumor xenografts without apparent adverse effects in SCID mice. Iodide induced an increase in the level of reactive oxygen species. Iodide-induced apoptosis is sensitive to N-acetylcysteine inhibition, suggesting an important role by reactive oxygen species in this apoptotic process. In addition, iodide-induced apoptosis is associated with overexpression of CDKN1A (p21/Waf1)and down-regulation of survivin at both mRNA and protein levels. This is the first report demonstrating that a therapeutic dose of nonradioactive iodide has potent efficacy and high selectivity against lung cancer when used in combination with genetic modification of cancer cells to express the NIS/TPO genes.

Cyclooxygenase-2-dependent expression of angiogenic CXC chemokines ENA-78/CXC Ligand (CXCL) 5 and interleukin-8/CXCL8 in human non-small cell lung cancer.

Elevated tumor cyclooxygenase (COX)-2 activity plays a multifaceted role in non-small cell lung cancer (NSCLC). To elucidate the role of COX-2 in the in vitro and in vivo expression of two known NSCLC angiogenic peptides, CXC ligand (CXCL) 8 and CXCL5, we studied two COX-2 gene-modified NSCLC cell lines, A549 and H157. COX-2 overexpression enhanced the in vitro expression of both CXCL8 and CXCL5. In contrast, specific COX-2 inhibition decreased the production of both peptides as well as nuclear translocation of nuclear factor kappaB. In a severe combined immunodeficient mouse model of human NSCLC, the enhanced tumor growth of COX-2-overexpressing tumors was inhibited by neutralizing anti-CXCL5 and anti-CXCL8 antisera. We conclude that COX-2 contributes to the progression of NSCLC tumorigenesis by enhancing the expression of angiogenic chemokines CXCL8 and CXCL5.

Methanandamide increases COX-2 expression and tumor growth in murine lung cancer.

Increased COX-2 expression and elevated PGE2 have been associated with a poor prognosis in lung cancer. Cannabinoids have been known to exert some of their biological effects via modulation of prostaglandin production. We evaluated the impact of methanandamide on COX-2 expression, PGE2 production, and tumor growth in murine lung cancer. Methanandamide administration (5 mg/kg, four times/wk i.p.) resulted in an increased rate of tumor growth (P<0.01 compared with diluent treated controls). The CB1 and CB2 receptor antagonists, SR141716 and SR144528, did not block the methanandamide-mediated increase in tumor growth. In vivo, methanandamide treatment increased the production of PGE2 at the tumor site as well as in splenocytes. Consistent with these results, methanandamide increased PGE2 and COX-2 levels in murine lung cancer cells in vitro via a cannabinoid receptor-independent mechanism. The COX-2-specific inhibitor, SC58236, abrogated methanandamide induction of PGE2 production in vitro and blocked methanandamide-enhanced tumor growth in vivo. Furthermore, the p38/MAPK inhibitor, SB203528, and the p42/44 inhibitor, PD98059, blocked methanandamide-mediated induction of PGE2 and COX-2. These results suggest that methanandamide augments tumor growth by a cannabinoid receptor-independent pathway that is associated with the up-regulation of COX-2.

The effect of cyclooxygenase-2 (COX-2) inhibition on human prostate cancer induced osteoblastic and osteolytic lesions in bone.

BACKGROUND: The mechanism of bone formation by osteoblastic prostate cancer metastases is not well defined. Using knockout mice, it has been demonstrated that prostaglandins produced by COX-2 are critical for fracture repair. Therefore, our aim was to determine if COX-2 plays a role in the bone formation in osteoblastic prostate cancer metastases in bone. MATERIALS AND METHODS: We assessed the influence of pharmacologic COX-2 inhibition in a SCID mouse intratibial injection model of bone metastasis using two human prostate cancer cell lines that produce either osteoblastic lesions (LAPC-9) or osteolytic lesions (PC-3, negative control). SC-58236, a COX-2 specific inhibitor, was used at a dose of 3 mg/Kg intraperitoneally 3 times per week in the Treatment groups for 8 weeks until sacrifice. RESULTS: Western blot for COX-2 demonstrated that LAPC-9 cells expressed high levels of COX-2 while PC-3 cells did not. Treatment with SC-58236 significantly reduced the size of osteoblastic lesions after LAPC-9 injection based on both radiographic and histomorphometric criteria compared to the control group. In contrast, large osteolytic lesions were seen in both control and SC-58236 treated animals after PC-3 cell injections. The results of this study indicate that COX-2 inhibition can decrease the size of osteoblastic lesions produced by LAPC-9, a human prostate cancer cell line that expresses high levels of COX-2. This treatment had no effect on the osteolytic activity of PC-3 cells. CONCLUSION: These findings suggest that the progression of osteoblastic metastases induced by human prostate cancer cells may be limited by COX-2 inhibitors.

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.

SLC/CCL21-mediated anti-tumor responses require IFNgamma, MIG/CXCL9 and IP-10/CXCL10.

BACKGROUND: SLC/CCL21, normally expressed in high endothelial venules and in T cell zones of spleen and lymph nodes, strongly attracts T cells and dendritic cells (DC). We have previously shown that SLC/CCL21-mediated anti-tumor responses are accompanied by significant induction of IFNgamma and the CXC chemokines, monokine induced by IFNgamma (MIG/CXCL9) and IFNgamma-inducible protein-10 (IP-10/CXCL10). RESULTS: We assessed the importance of IFNgamma, IP-10/CXCL10 and MIG/CXCL9 in SLC/CCL21 therapy. In vivo depletion of IP-10/CXCL10, MIG/CXCL9 or IFNgamma significantly reduced the anti-tumor efficacy of SLC/CCL21. Assessment of cytokine production at the tumor site showed an interdependence of IFNgamma, MIG/CXCL9 and IP-10/CXCL10; neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines. Similarly, neutralization of any one of these cytokines led to a decrease in the frequency of CXCR3+ve T cells and CD11c+ve DC at the tumor site. CONCLUSION: These findings indicate that the full potency of SLC/CCL21-mediated anti-tumor responses require in part the induction of IFNgamma, MIG/CXCL9 and IP-10/CXCL10.

Cocaine modulates cytokine and enhances tumor growth through sigma receptors.

Sigma receptors are intracellular receptors that interact with a variety of psychotropic ligands, including cocaine. Administration of cocaine to mice promoted the in vivo growth of a syngeneic lung cancer cell line and identical effects were observed with PRE 084, a selective sigma(1) receptor agonist. Increased tumor growth was accompanied by an increase in IL-10 and a decrease in IFN-gamma production in splenocytes and at the tumor site. The tumor-promoting effects produced by both cocaine and PRE 084 were abrogated by administration of specific antibodies to IL-10, or by administration of a sigma(1) receptor antagonist. We conclude that sigma(1) receptor ligands, including cocaine, augment tumor growth via a cytokine-dependent, receptor-mediated mechanism that involves regulation of T helper 1/T helper 2 cytokine balance.

Snail promotes CXCR2 ligand-dependent tumor progression in non-small cell lung carcinoma.

PURPOSE: As a transcriptional repressor of E-cadherin, Snail has predominantly been associated with epithelial-mesenchymal transition, invasion, and metastasis. However, other important Snail-dependent malignant phenotypes have not been fully explored. Here, we investigate the contributions of Snail to the progression of non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Immunohistochemistry was done to quantify and localize Snail in human lung cancer tissues, and tissue microarray analysis was used to correlate these findings with survival. NSCLC cell lines gene-modified to stably overexpress Snail were evaluated in vivo in two severe combined immunodeficiency murine tumor models. Differential gene expression between Snail-overexpressing and control cell lines was evaluated using gene expression microarray analysis. RESULTS: Snail is upregulated in human NSCLC tissue, and high levels of Snail expression correlate with decreased survival (P < 0.026). In a heterotopic model, mice bearing Snail-overexpressing tumors developed increased primary tumor burden (P = 0.008). In an orthotopic model, mice bearing Snail-overexpressing tumors also showed a trend toward increased metastases. In addition, Snail overexpression led to increased angiogenesis in primary tumors as measured by MECA-32 (P < 0.05) positivity and CXCL8 (P = 0.002) and CXCL5 (P = 0.0003) concentrations in tumor homogenates. Demonstrating the importance of these proangiogenic chemokines, the Snail-mediated increase in tumor burden was abrogated with CXCR2 blockade. Gene expression analysis also revealed Snail-associated differential gene expression with the potential to affect angiogenesis and diverse aspects of lung cancer progression. CONCLUSION: Snail upregulation plays a role in human NSCLC by promoting tumor progression mediated by CXCR2 ligands.

IL-10 mediates sigma 1 receptor-dependent suppression of antitumor immunity.

Sigma receptors are unique endoplasmic reticulum proteins that mediate signaling for a variety of drugs. We determined the effect of sigma(1) receptor agonists on immune responses in a syngeneic lung cancer model. Sigma(1) receptor agonists, including cocaine, up-regulated splenocyte IL-10 mRNA and protein production in vitro in a sigma receptor-dependent, pertussis toxin-sensitive manner. In vivo, sigma(1) receptor agonists promoted tumor growth and induced IL-10 at the tumor site. Increased tumor growth was prevented by administration of specific Abs to IL-10 or by administration of specific sigma(1) receptor antagonists. We report that sigma(1) receptor ligands, including cocaine, augment tumor growth through an IL-10 dependent mechanism.

Interleukin-7 and transforming growth factor-beta play counter-regulatory roles in protein kinase C-delta-dependent control of fibroblast collagen synthesis in pulmonary fibrosis.

Transforming growth factor-beta (TGF-beta) is a potent fibrogenic factor responsible for promoting synthesis of extracellular matrix. Interleukin-7 (IL-7) inhibits TGF-beta signaling by up-regulating Smad7, a major inhibitor of the Smad family. In a variety of cells, TGF-beta-mediated activation of target genes requires active protein kinase C-delta (PKC-delta) in addition to Smads (1). We determined the role of PKC-delta in the regulation of pulmonary fibroblast collagen synthesis in response to TGF-beta and IL-7 stimulation. Here we show that TGF-beta and IL-7 have opposing effects on PKC-delta; TGF-beta stimulates, while IL-7 inhibits, PKC-delta activity. IL-7 inhibits TGF-beta-induced PKC-delta phosphorylation at Ser-645 and Thr-505. Inhibition of PKC-delta with specific small inhibitory RNA restores TGF-beta-mediated induction of Smad7 and in parallel significantly reduces TGF-beta-mediated collagen synthesis. Thus, PKC-delta may play a critical role in the pathogenesis of pulmonary fibrosis and may serve as a molecular target for therapeutic intervention to suppress fibrosis.