Robert A. Winn

γ-Catenin expression is reduced or absent in a subset of human lung cancers and re-expression inhibits transformed cell growth

Lung cancer is a heterogeneous disease categorized into multiple subtypes of cancers which likely arise from distinct patterns of genetic alterations and disruptions. Precedent exists for a role of β-catenin, a downstream component of the Wnt signaling pathway that serves as a transcriptional co-activator with TCF/LEF, in several human cancers including colon carcinomas. In this study, we observed that β-catenin was highly and uniformly expressed in a panel of NSCLC cell lines and primary lung tumors. By contrast, γ-catenin was weakly expressed or absent in several NSCLC cell lines and immunohistochemical analysis of primary NSCLC tumors revealed negligible to weak γ-catenin staining in ∼30% of the specimens. Treatment of NSCLC cells expressing reduced γ-catenin protein with 5-aza-2′-deoxycytidine (5aza2dc), a DNA methylation inhibitor, or trichostatin A (TSA), a histone deacetylase inhibitor, increased γ-catenin protein content in NSCLC cells with low γ-catenin expression. Significantly, the activity of a β-catenin/TCF-dependent luciferase reporter was markedly elevated in the NSCLC cell lines that underexpressed γ-catenin relative to those lines that highly expressed γ-catenin. Moreover, transfection of these cells with a γ-catenin expression plasmid reduced the elevated TCF activity by 85% and strongly inhibited cell growth on tissue culture plastic as well as anchorage-independent growth in soft agar. This study shows that γ-catenin can function as an inhibitor of β-catenin/TCF-dependent gene transcription and highlights γ-catenin as a potentially novel tumor suppressor protein in a subset of human NSCLC cancers.

Peroxisome proliferator-activated receptor-γ (PPARγ) inhibits tumorigenesis by reversing the undifferentiated phenotype of metastatic non-small-cell lung cancer cells (NSCLC)

Pharmacological activators of peroxisome proliferator-activated receptor-γ (PPARγ) have been shown to inhibit growth of lung tumors largely through growth inhibition and induction of apopotosis. However, since many of these agents engage other effectors, the role of PPARγ in lung tumorigenesis remains poorly defined. To specifically examine PPARγ-mediated events, non-small-cell lung cancer (NSCLC) cells overexpressing PPARγ were established. Overexpression of PPARγ in H2122 adenocarcinoma cells (H2122-PPARγ) blocked anchorage-independent growth compared to cells transfected with empty vector (H2122-LNCX), but had no significant effect on cell proliferation or apoptosis under standard tissue culture conditions. Orthotopic implantation of H2122-PPARγ cells into the lungs of nude rats inhibited tumor growth and metastasis in vivo and prolonged survival compared to implantation of H2122-LNCX cells. Consistent with these findings, H2122-PPARγ cells had an impaired invasiveness as assessed in Transwell assays. In a three-dimensional culture system, H2122-PPARγ cells formed polarized spheroid structures similar to those observed with normal lung epithelial cells. H2122-LNCX cells formed nonpolarized aggregate structures and did not show any of these epithelial properties. These data indicate that inhibitory effects of PPARγ on lung tumorigenesis involve selective inhibition of invasive metastasis, and activation of pathways that promote a more differentiated epithelial phenotype.

Antitumorigenic Effect of Wnt 7a and Fzd 9 in Non-small Cell Lung Cancer Cells Is Mediated through ERK-5-dependent Activation of Peroxisome Proliferator-activated Receptor γ

The Wnt pathway is critical for normal development, and mutation of specific components is seen in carcinomas of diverse origins. The role of this pathway in lung tumorigenesis has not been clearly established. Recent studies from our laboratory indicate that combined expression of the combination of Wnt 7a and Frizzled 9 (Fzd 9) in Non-small Cell Lung Cancer (NSCLC) cell lines inhibits transformed growth. We have also shown that increased expression of peroxisome proliferator-activated receptor γ (PPARγ) inhibits transformed growth of NSCLC and promotes epithelial differentiation of these cells. The goal of this study was to determine whether the effects of Wnt 7a/Fzd 9 were mediated through PPARγ. We found that Wnt 7a and Fzd 9 expression led to increased PPARγ activity. This effect was not mediated by altered expression of the protein. Wnt 7a and Fzd 9 expression resulted in activation of ERK5, which was required for PPARγ activation in NSCLC. SR 202, a known PPARγ inhibitor, blocked the increase in PPARγ activity and restored anchorage-independent growth in NSCLC expressing Wnt 7a and Fzd 9. SR 202 also reversed the increase in E-cadherin expression mediated by Wnt 7a and Fzd 9. These data suggest that ERK5-dependent activation of PPARγ represents a major effector pathway mediating the anti-tumorigenic effects of Wnt 7a and Fzd 9 in NSCLC.

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

Prostacyclin Prevents Murine Lung Cancer Independent of the Membrane Receptor by Activation of Peroxisomal Proliferator–Activated Receptor γ

Overexpression of prostacyclin synthase (PGIS) decreases lung tumor multiplicity in chemical- and cigarette-smoke–induced murine lung cancer models. Prostacyclin signals through a single G-protein–coupled receptor (IP), which signals through cyclic AMP. To determine the role of this receptor in lung cancer chemoprevention by prostacyclin, PGIS-overexpressing mice were crossed to mice that lack the IP receptor [IP(−/−)]. Carcinogen-induced lung tumor incidence was similar in IP(+/+), IP(+/−), and IP(−/−) mice, and overexpression of PGIS gave equal protection in all three groups, indicating that the protective effects of prostacyclin are not mediated through activation of IP. Because prostacyclin can activate members of the peroxisomal proliferator–activated receptor (PPAR) family of nuclear receptors, we examined the role of PPARγ in the protection of prostacyclin against lung tumorigenesis. Iloprost, a stable prostacyclin analogue, activated PPARγ in nontransformed bronchial epithelial cells and in a subset of human non–small-cell lung cancer cell lines. Iloprost-impregnated chow fed to wild-type mice resulted in elevated lung macrophages and decreased lung tumor formation. Transgenic animals with lung-specific PPARγ overexpression also developed fewer lung tumors. This reduction was not enhanced by administration of supplemental iloprost. These studies indicate that PPARγ is a critical target for prostacyclin-mediated lung cancer chemoprevention and may also have therapeutic activity.

Wnt signaling pathway and lung disease.

The Wnt pathway plays an important role in development and in regulating adult stem cell systems. A variety of cellular processes is mediated by Wnt signaling, which includes cellular proliferation, differentiation, survival, apoptosis, and cell motility. Loss of regulation of these pathways can lead to tumorigenesis, and the Wnt pathway has been implicated in the development of several types of cancers, including colon, lung, leukemia, breast, thyroid, and prostate. The Wnt pathway has also been associated with other lung diseases such as interstitial lung disease (ILD) and asthma. Our increasing understanding of the Wnt pathway offers great hope that new molecular-based screening tests and pharmaceutical agents that selectively target this pathway will be developed to diagnose and treat these diseases in the future.

Mycoplasma pneumoniae-Associated Bronchiolitis Causing Severe Restrictive Lung Disease in Adults: Report of Three Cases and Literature Review

Study objectives To characterize adult Mycoplasma pneumoniae-induced bronchiolitis requiring hospitalization. Design We encountered an adult patient with severe bronchiolitis in the absence of pneumonia due to M pneumoniae. To determine the relative frequency of such a condition, we retrospectively reviewed the medical records of adults over a 4-year period with a hospital discharge diagnosis of “bronchiolitis” from a university hospital. Setting University Hospital of the University of Colorado Health Sciences Center, Denver, CO. Study subjects From 1994 to 1998, 10 adult inpatients were identified with a diagnosis of bronchiolitis. There were two with respiratory bronchiolitis, one with panbronchiolitis, one patient with bronchiolitis obliterans organizing pneumonia (BOOP), and six with acute inflammatory bronchiolitis. Including the initial patient, three had a definitive clinical diagnosis of Mycoplasma-associated bronchiolitis. Results The three adult patients with bronchiolitis due to M pneumoniae are unusual because they occurred in the absence of radiographic features of a lobar or patchy alveolar pneumonia. Hospital admission was occasioned by the severity of symptoms and gas exchange abnormalities. One patient had bronchiolitis as well as organizing pneumonia (BOOP) that responded favorably to corticosteroid treatment. The other two had high-resolution CT findings diagnostic of an acute inflammatory bronchiolitis. One of the patients with inflammatory bronchiolitis had an unusual pattern of marked ventilation and perfusion defects localized predominantly to the left lung. All three had restrictive ventilatory impairment on physiologic testing. Conclusions In adults, Mycoplasma-associated bronchiolitis without pneumonia is rarely reported, but in hospitalized patients, it may be more common than expected and may be associated with severe physiologic disturbances.

Antitumorigenic Effects of Peroxisome Proliferator-Activated Receptor-γ in Non-Small-Cell Lung Cancer Cells Are Mediated by Suppression of Cyclooxygenase-2 via Inhibition of Nuclear Factor-κB

Pharmacological activators of peroxisome proliferator-activated receptor-γ (PPARγ) inhibit growth of non-small-cell lung cancer (NSCLC) cell lines in vitro and in xenograft models. Because these agents engage off-target pathways, we have assessed the effects of PPARγ by overexpressing the protein in NSCLC cells. We reported previously that increased PPARγ inhibits transformed growth and invasiveness and promotes epithelial differentiation in a panel of NSCLC expressing oncogenic K-Ras. These cells express high levels of cyclooxygenase-2 (COX-2) and produce high levels of prostaglandin E2 (PGE2). The goal of these studies was to identify the molecular mechanisms whereby PPARγ inhibits tumorigenesis. Increased PPARγ inhibited expression of COX-2 protein and promoter activity, resulting in decreased PGE2 production. Suppression of COX-2 was mediated through increased activity of the tumor suppressor phosphatase and tensin homolog, leading to decreased levels of phospho-Akt and inhibition of nuclear factor-κB activity. Pharmacological inhibition of PGE2 production mimicked the effects of PPARγ on epithelial differentiation in three-dimensional culture, and exogenous PGE2 reversed the effects of increased PPARγ activity. Transgenic mice overexpressing PPARγ under the control of the surfactant protein C promoter had reduced expression of COX-2 in type II cells and were protected against developing lung tumors in a chemical carcinogenesis model. These data indicate that high levels of PGE2 as a result of elevated COX-2 expression are critical for promoting lung tumorigenesis and that the antitumorigenic effects of PPARγ are mediated in part through blocking this pathway.

Inhibited Neurogenesis in JNK1-Deficient Embryonic Stem Cells

ABSTRACT The JNKs are components of stress signaling pathways but also regulate morphogenesis and differentiation. Previously, we invoked a role for the JNKs in nerve growth factor (NGF)-stimulated PC12 cell neural differentiation (L. Marek et al., J. Cell. Physiol. 201:459-469, 2004; E. Zentrich et al., J. Biol. Chem. 277:4110-4118, 2002). Herein, the role for JNKs in neural differentiation and transcriptional regulation of the marker gene, NFLC, modeled in mouse embryonic stem (ES) cells was studied. NFLC-luciferase reporters revealed the requirement for NFLC promoter sequences encompassing base pairs −128 to −98 relative to the transcriptional start site as well as a proximal cyclic AMP response element-activating transcription factor binding site at −45 to −38 base pairs for transcriptional induction in NGF-treated PC12 cells and neurally differentiated ES cells. The findings reveal common promoter sequences that integrate conserved signal pathways in both PC12 cell and ES cell systems. To test the requirement for the JNK pathway in ES cell neurogenesis, ES cell lines bearing homozygous disruptions of the jnk1, jnk2, or jnk3 genes were derived and submitted to an embryoid body (EB) differentiation protocol. Neural differentiation was observed in wild-type, JNK2−/−, and JNK3−/− cultures but not in JNK1−/− EBs. Rather, an outgrowth of cells with epithelial morphology and enhanced E-cadherin expression but low NFLC mRNA and protein was observed in JNK1−/− cultures. The expression of wnt-4 and wnt-6, identified inhibitors of ES cell neurogenesis, was significantly elevated in JNK1−/− cultures relative to wild-type, JNK2−/−, and JNK3−/− cultures. Moreover, the Wnt antagonist, sFRP-2, partially rescued neural differentiation in JNK1−/− cultures. Thus, a genetic approach using JNK-deficient ES cells reveals a novel role for JNK1 involving repression of Wnt expression in neural differentiation modeled in murine ES cells.

Depletion of Cytosolic Phospholipase A2 in Bone Marrow-Derived Macrophages Protects against Lung Cancer Progression and Metastasis

Cancer progression and metastasis involves interactions between tumor cells and the tumor microenvironment (TME). We reported that mice deficient for cytosolic phospholipase A(2) (cPLA(2)-KO) are protected against the development of lung tumors. The goal of this study was to examine the role of cPLA(2) in the TME. Mouse lung cancer cells (CMT167 and Lewis lung carcinoma cells) injected directly into lungs of syngeneic mice formed a primary tumor, and then metastasized to other lobes of the lung and to the mediastinal lymph nodes. Identical cells injected into cPLA(2)-KO mice showed a dramatic decrease in the numbers of secondary metastatic tumors. This was associated with decreased macrophage staining surrounding the tumor. Wild-type mice transplanted with cPLA(2)-KO bone marrow had a marked survival advantage after inoculation with tumor cells compared with mice receiving wild-type (WT) bone marrow. In vitro, coculturing CMT167 cells with bone marrow-derived macrophages from WT mice increased production of interleukin 6 (IL-6) by cancer cells. This increase was blocked in cocultures using cPLA(2)-KO macrophages. Correspondingly, IL-6 staining was decreased in tumors grown in cPLA(2)-KO mice. These data suggest that stromal cPLA(2) plays a critical role in tumor progression by altering tumor-macrophage interactions and cytokine production.