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.

Background: PRMT1 is up-regulated in lung cancer.Results: PRMT1 is a novel regulator of EMT and Twist1 is a new PRMT1 substrate.Conclusion: PRMT1-methylation of Twist1 is required for active E-cadherin repression.Significance: Targeting PRMT1-mediated Twist1 methylation might represent a novel strategy for developing new anti-invasive/anti-metastatic drugs.

Cellular senescence is an initial barrier for carcinogenesis. However, the signaling mechanisms that trigger cellular senescence are incompletely understood, particularly in vivo. Here we identify Wnt7a as a novel upstream inducer of cellular senescence. In two different mouse strains (C57Bl/6J and FVB/NJ), we show that the loss of Wnt7a is a major contributing factor for increased lung tumorigenesis owing to reduced cellular senescence, and not reduced apoptosis, or autophagy. Wnt7a-null mice under de novo conditions and in both the strains display E-cadherin-to-N-cadherin switch, reduced expression of cellular senescence markers and reduced expression of senescence-associated secretory phenotype, indicating a genetic predisposition of these mice to increased carcinogen-induced lung tumorigenesis. Interestingly, Wnt7a induced an alternate senescence pathway, which was independent of β-catenin, and distinct from that of classical oncogene-induced senescence mediated by the well-known p16INK4a and p19ARF pathways. Mechanistically, Wnt7a induced cellular senescence via inactivation of S-phase kinase-associated protein 2, an important alternate regulator of cellular senescence. Additionally, we identified Iloprost, a prostacyclin analog, which initiates downstream signaling cascades similar to that of Wnt7a, as a novel inducer of cellular senescence, presenting potential future clinical translational strategies. Thus pro-senescence therapies using either Wnt7a or its mimic, Iloprost, might represent a new class of therapeutic treatments for lung cancer.

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.

Background: γ-Catenin is a tumor suppressor in lung cancer.Results: γ-Catenin is a novel regulator of HAI-1 and cell migration.Conclusion: γ-Catenin and HAI-1 regulate cell migration and sensitize NSCLC cells to c-MET inhibitor.Significance: Targeting γ-catenin-mediated HAI-1 expression might be a new strategy to treat lung cancer.

The authors wish to make readers aware of a typographical error in the above paper for the amount of urethane administered to the Wnt7a mice. The amount given should read as 1 mg/g body weight not 1 mg/kg body weight.In the Results section in page 4, the following paragraph should be amended to read:To test this idea, we probed for the effects of urethane (ethyl carbamate)-induced lung tumor formation in wild type and Wnt7a-null mice, a prototypical model to study lung tumorigenesis. [26][27][28] Urethane is a chemical carcinogen, which causes activating mutations in K-Ras, 27,28 leading to the formation of lung tumors in mice. Several studies have highlighted a strainspecific response to urethane; for example, FVB/NJ strains are susceptible to lung tumorigenesis, whereas C57Bl/6J, in contrast, are more resistant. [26][27][28] Therefore, in our studies, wild type and Wnt7a null in FVB/NJ mice received the standard single dose of 1 mg/g body weight of urethane, whereas wild type and Wnt7a null in C57Bl/6J mice received weekly injections of 1 mg/g body weight of urethane for 6 weeks. The mice were euthanized and dissected after 20 weeks (FVB/NJ strains) or 40 weeks (C57Bl/6J strains) to assess the formation of lung tumors.In the Materials and Methods section on page 10, the following paragraph should be amended to read:Urethane treatment. Six-to-eight-week-old mice were given intraperitoneal injections of either 0.9% saline or urethane (1 mg/g body weight). Wild-type and Wnt7a-null mice in FVB/NJ strain received the standard single dose of urethane, whereas the wild-type and Wnt7a-null mice in C57Bl/6J strain received weekly injections of 1 mg/g body weight of urethane for 6 weeks. The control and experimental mice were weighed weekly to observe any changes in body weight until they were euthanized. The mice were euthanized and dissected after 20 weeks (FVB/NJ strains) or 40 weeks (C57Bl/6J strains) to assess the formation of lung tumors. Lung tumors were counted and measured using digital calipers (Fisher Scientific, Waltham, MA, USA).The authors apologise for any inconvenience caused by this error.This error has now been rectified, and the corrected article appears in this issue. The html and online pdf versions have also been rectified, and now carry the corrected paper.

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3 H] methionine). Here we describe a stepby-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of Sadenosyl-L-[methyl-3 H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation. Video LinkThe video component of this article can be found at

No abstract