The Wnt signaling pathway is critical in normal development, and mutation of specific components is frequently observed in carcinomas of diverse origins. However, the potential involvement of this pathway in lung tumorigenesis has not been established. In this study, analysis of multiple Wnt mRNAs in non-small cell lung cancer (NSCLC) cell lines and primary lung tumors revealed markedly decreased Wnt-7a expression compared with normal short-term bronchial epithelial cell lines and normal uninvolved lung tissue. Wnt-7a transfection in NSCLC cell lines reversed cellular transformation, decreased anchorage-independent growth, and induced epithelial differentiation as demonstrated by soft agar and three-dimensional cell culture assays in a subset of the NSCLC cell lines. The action of Wnt-7a correlated with expression of the specific Wnt receptor Frizzled-9 (Fzd-9), and transfection of Fzd-9 into a Wnt-7a-insensitive NSCLC cell line established Wnt-7a sensitivity. Moreover, Wnt-7a was present in Fzd-9 immunoprecipitates, indicating a direct interaction of Wnt-7a and Fzd-9. In NSCLC cells, Wnt-7a and Fzd-9 induced both cadherin and Sprouty-4 expression and stimulated the JNK pathway, but not ␤-catenin/T cell factor activity. In addition, transfection of gain-of-function JNK strongly inhibited anchorage-independent growth. Thus, this study demonstrates that Wnt-7a and Fzd-9 signaling through activation of the JNK pathway induces cadherin proteins and the receptor tyrosine kinase inhibitor Sprouty-4 and represents a novel tumor suppressor pathway in lung cancer that is required for maintenance of epithelial differentiation and inhibition of transformed cell growth in a subset of human NSCLCs.

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 ␥ (PPAR␥) 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 PPAR␥. We found that Wnt 7a and Fzd 9 expression led to increased PPAR␥ 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 PPAR␥ activation in NSCLC. SR 202, a known PPAR␥ inhibitor, blocked the increase in PPAR␥ 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 PPAR␥ represents a major effector pathway mediating the anti-tumorigenic effects of Wnt 7a and Fzd 9 in NSCLC.Wnts are a family of secreted glycoproteins that serve as extracellular signaling molecules controlling diverse morphogenic and developmental programs (1). Signaling is mediated by a family of distinct seven-membrane receptors known as Frizzled (Fzd) 2 (2), and is further regulated by co-receptors LRP 5/6 (3). Aberrant Wnt signaling has been implicated in a variety of cancers (4, 5). Our laboratory has focused on the role of Wnt signaling in lung cancer. We have previously reported that the restoration of Wnt 7a and Fzd 9 signaling inhibited both cell proliferation and anchorage-independent growth, promoted cellular differentiation, and reversed the transformed phenotype in Non-small Cell Lung Cancer cells (NSCLC) (6). These findings unveil a novel tumor suppressor pathway in lung cancer and implicate Wnt 7a and Fzd 9 in the maintenance of epithelial cellular differentiation. The downstream effector pathways mediating these effects are not well understood. Proliferator-activated receptor ␥ (PPAR␥) is a member of the PPAR family of ligand-activated nuclear receptors implicated in a wide variety of biological functions (7). Three PPAR isoforms have been identified, ␣, ␥, and ␤/␦, which all bind as heterodimers with the retinoic acid X receptor to specific regulatory elements in the promoter regions of their target genes. The role of PPAR␥ has been extensively studied in a variety of cancers including colon, breast, prostate, and lung (see Ref. 8 for review). Inactivating mutations in the PPAR␥ gene have been seen in colon cancers (9, 10), suggesting that PPAR␥ behaves as a tumor suppressor gene. Pharmacological activators of PPAR␥ inhibit growth of NSCLC cells and induce ap...

Purpose Lung adenocarcinoma (AdC) and lung squamous cell carcinoma (SCC) are the most common non-small cell lung cancer (NSCLC) subtypes. This study was designed to determine whether reduced expression of transforming growth factor β type II receptor (TGFβRII) promotes lung AdC and SCC carcinogenesis. Experimental Design We examined TGFβRII expression at the protein and mRNA levels in human NSCLC samples and assessed the relationship between TGFβRII expression and clinico-pathologic parameters. To determine if sporadic TGFβRII deletion in airway epithelial cells induces NSCLC formation, we targeted TGFβRII deletion alone and in combination with oncogenic KrasG12D to murine airways using a keratin 5 (K5) promoter and inducible Cre recombinase. Results Reduced TGFβRII expression in human NSCLC is associated with male gender, smoking, SCC histology, reduced differentiation, increased tumor stage, increased nodal metastasis, and reduced survival. Homozygous or heterozygous TGFβRII deletion in mouse airway epithelia increases the size and number of KrasG12D-initiated AdC and SCC. TGFβRII deletion increases proliferation, local inflammation, and TGFβ ligand elaboration; TGFβRII knockdown in airway epithelial cells increases migration and invasion. Conclusions Reduced TGFβRII expression in human NSCLC is associated with more aggressive tumor behavior and inflammation that is at least partially mediated by increased TGFβ1 expression. TGFβRII deletion in mouse airway epithelial cells promotes AdC and SCC formation, indicating that TGFβRII loss plays a causal role in lung carcinogenesis. That TGFβRII demonstrates haploid insufficiency, suggests that a 50% TGFβRII protein reduction would negatively impact lung cancer prognosis.

Lung adenocarcinoma (AdC) and lung squamous cell carcinoma (SCC) are the most common non-small cell lung cancer (NSCLC) subtypes, however, most genetic mouse models of lung cancer produce predominantly, if not exclusively, AdC. Whether this is secondary to targeting mutations to the distal airway cells or to the use of activating Kras mutations that drive AdC formation is unknown. We previously showed that targeting KrasG12D activation and transforming growth factor β receptor type II (TGFβRII) deletion to airway basal cells via a keratin promoter induced formation of both lung AdC and SCC. In this study we assessed if targeting phosphatase and tensin homologue (PTEN) deletion to airway basal cells could initiate lung tumor formation or increase lung SCC formation. We found that PTEN deletion is capable of initiating both lung AdC and SCC formation when targeted to basal cells and although PTEN deletion is a weaker tumor initiator than KrasG12D with low tumor multiplicity and long latency, tumors initiated by PTEN deletion were larger and displayed more malignant conversion than KrasG12D initiated tumors. That PTEN deletion did not increase lung SCC formation compared to KrasG12D activation, suggests that the initiating genetic event does not dictate tumor histology when genetic alterations are targeted to a specific cell. These studies also confirm that basal cells of the conducting airway are capable of giving rise to multiple NSCLC tumor types.