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.

This study aimed to (a) determine if DNA methylation is a mechanism of WWOX (WW domain containing oxidoreductase) and FHIT (fragile histidine triad) inactivation in lung, breast and bladder cancers; (b) examine distinct methylation patterns in neoplastic and adjacent tissues and (c) seek correlation of methylation patterns with disease status. Protein expression was detected by immunohistochemistry, and methylation status by methylation-specific PCR (MSP) and sequencing, in lung squamous cell carcinomas and adjacent tissues, invasive breast carcinomas, adjacent tissues and normal mammary tissues and bladder transitional cell carcinomas. Wwox and Fhit expression was reduced in cancers in association with hypermethylation. Differential patterns of WWOX and FHIT methylation were observed in neoplastic vs adjacent non-neoplastic tissues, suggesting that targeted MSP amplification could be useful in following treatment or prevention protocols. WWOX promoter MSP differentiates DNA of lung cancer from DNA of adjacent lung tissue. WWOX and FHIT promoter methylation is detected in tissue adjacent to breast cancer and WWOX exon 1 MSP distinguishes breast cancer DNA from DNA of adjacent and normal tissue. Differential methylation in cancerous vs adjacent tissues suggests that WWOX and FHIT hypermethylation analyses could enrich a panel of DNA methylation markers.

BACKGROUND FHIT and WWOX are a tumor suppressor and a candidate suppressor that encompass the FRA3B and FRA16D fragile sites at chromosomes 3p14.2 and 16q23.3–24.1, respectively. Reduced or absent Fhit expression has been reported in two‐thirds of invasive breast tumors in association with adverse prognostic factors. Loss of 16q has been reported frequently in low‐grade, invasive breast tumors. METHODS Expression of Fhit and Wwox was evaluated by immunohistochemical staining in 97 archived breast carcinoma specimens. Expression levels were analyzed for correlations with each other, as well as with various patient and tumor characteristics. RESULTS Reduced Fhit and Wwox expression in tumors was observed in 54.6% and 63.2% of specimens, respectively. Fhit and Wwox expression were correlated strongly (P = 0.001). Reduced Fhit staining was seen more frequently in premenopausal patients (P = 0.010), estrogen receptor (ER)‐negative or scantly ER‐positive tumors (P = 0.058 borderline), high‐grade tumors (P = 0.005), and tumors with metastases (P = 0.041). Reduced Wwox staining was more common in tumors with less favorable ER status (P = 0.033). Wwox expression in normal tissue was reduced in 32.9% of specimens, especially in patients with higher stage disease (P = 0.033). Severely reduced Wwox staining (extent < 10%) in normal tissue was found only in postmenopausal women, but reduced Wwox staining (11–75%) was more common in premenopausal women (P = 0.012). Tumor status, lymph node status, and intensity of Fhit expression in tumors were related independently to survival (P = 0.003, P < 0.001, and P = 0.046, respectively). CONCLUSIONS The strong correlation observed between Fhit and Wwox expression was consistent with the common elevated susceptibility of fragile sites to DNA damage. Reduced Fhit expression is associated with adverse prognostic factors. The current results suggest that Wwox also has an important and complex association with steroid hormone expression and breast carcinogenesis. Cancer 2004. © 2004 American Cancer Society.

The effects of the phytohormone abscisic acid (ABA) on plant growth and root nodule formation were analyzed in Trifolium repense (white clover) and Lotus japonicus, which form indeterminate and determinate nodules, respectively. In T. repense, although the number of nodules formed after inoculation with Rhizobium leguminosarum bv. trifolii strain 4S (wild type) was slightly affected by exogenous ABA, those formed by strain H1(pC4S8), which forms ineffective nodules, were dramatically reduced 28 days after inoculation (DAI). At 14 and 21 DAI, the number of nodules formed with the wild-type strain was decreased by exogenous ABA. In L. japonicus, the number of nodules was also reduced by ABA treatment. Thus, exogenous ABA inhibits root nodule formation after inoculation with rhizobia. Observation of root hair deformation revealed that ABA blocked the step between root hair swelling and curling. When the ABA concentration in plants was decreased by using abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, the number of nodules on lateral roots of abamine-treated L. japonicus increased dramatically, indicating that lower-than-normal concentrations of endogenous ABA enhance nodule formation. We hypothesize that the ABA concentration controls the number of root nodules.