Sreedevi Avasarala

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.

PRMT1 Is a Novel Regulator of Epithelial-Mesenchymal-Transition in Non-small Cell Lung Cancer

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. Protein arginine methyl transferase 1 (PRMT1) was shown to be up-regulated in cancers and important for cancer cell proliferation. However, the role of PRMT1 in lung cancer progression and metastasis remains incompletely understood. In the present study, we show that PRMT1 is an important regulator of epithelial-mesenchymal transition (EMT), cancer cell migration, and invasion, which are essential processes during cancer progression, and metastasis. Additionally, we have identified Twist1, a basic helix-loop-helix transcription factor and a well-known E-cadherin repressor, as a novel PRMT1 substrate. Taken together, we show that PRMT1 is a novel regulator of EMT and arginine 34 (Arg-34) methylation of Twist1 as a unique “methyl arginine mark” for active E-cadherin repression. Therefore, targeting PRMT1-mediated Twist1 methylation might represent a novel strategy for developing new anti-invasive/anti-metastatic drugs. Moreover, methylated Twist1 (Arg-34), as such, could also emerge as a potential important biomarker for lung cancer.

Non-small-cell lung cancer: molecular targeted therapy and personalized medicine - drug resistance, mechanisms, and strategies

Targeted therapies for cancer bring the hope of specific treatment, providing high efficacy and in some cases lower toxicity than conventional treatment. Although targeted therapeutics have helped immensely in the treatment of several cancers, like chronic myelogenous leukemia, colon cancer, and breast cancer, the benefit of these agents in the treatment of lung cancer remains limited, in part due to the development of drug resistance. In this review, we discuss the mechanisms of drug resistance and the current strategies used to treat lung cancer. A better understanding of these drug-resistance mechanisms could potentially benefit from the development of a more robust personalized medicine approach for the treatment of lung cancer.

Dishevelled3 is a novel arginine methyl transferase substrate

Dishevelled, a phosphoprotein scaffold, is a central component in all the Wnt-sensitive signaling pathways. In the present study, we report that Dishevelled is post-translationally modified, both in vitro and in vivo, via arginine methylation. We also show protein arginine methyl transferases 1 and 7 as the key enzymes catalyzing Dishevelled methylation. Interestingly, Wnt3a stimulation of F9 teratocarcinoma cells results in reduced Dishevelled methylation. Similarly, the methylation-deficient mutant of Dishevelled, R271K, displayed spontaneous membrane localization and robust activation of Wnt signaling; suggesting that differential methylation of Dishevelled plays an important role in Wnt signaling. Thus arginine methylation is shown to be an important switch in regulation of Dishevelled function and Wnt signaling.

Wnt7a is a novel inducer of β-catenin-independent tumor-suppressive cellular senescence in lung cancer.

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.

Novel Role for γ-Catenin in the Regulation of Cancer Cell Migration via the Induction of Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1).

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. γ-catenin (Plakoglobin), a well-described structural protein functioning at the adherens junctions and desmosomes, was shown to be either lost or weakly expressed in non-small cell lung cancer (NSCLC) cells and tumor tissues. However, the tumor suppressive affects of γ-catenin were not fully understood. In this study, we have identified a novel role for the affects of γ-catenin on non-small cell lung cancer (NSCLC) cell migration. Expression of γ-catenin in NSCLC cells resulted in reduced cell migration as determined by both scratch assays and trans-well cell migration assays. Moreover, the affects of γ-catenin on cell migration were observed to be p53-dependent. Mechanistically, the anti-migratory effects seen via γ-catenin were driven by the expression of hepatocyte growth factor activator inhibitor Type I (HAI-1 or SPINT-1), an upstream inhibitor of the c-MET signaling pathway. Furthermore, the re-expression of γ-catenin sensitized NSCLC cells to c-MET inhibitor-mediated growth inhibition. Taken together, we identify γ-catenin as a novel regulator of HAI-1, which is a critical regulator of HGF/c-MET signaling. Therefore, targeting γ-catenin-mediated HAI-1 expression might be a useful strategy to sensitize NSCLC to c-MET inhibitors.

hsa-miR29b, a critical downstream target of non-canonical Wnt signaling, plays an anti-proliferative role in non-small cell lung cancer cells via targeting MDM2 expression.

Summary In non-small cell lung cancer cell lines, activation of &bgr;-catenin independent signaling, via Wnt7a/Frizzled9 signaling, leads to reversal of cellular transformation, reduced anchorage-independent growth and induction of epithelial differentiation. miRNA expression profiling on a human lung adenocarcinoma cell line (A549) identified hsa-miR29b as an important downstream target of Wnt7a/Frizzled9 signaling. We show herein that hsa-miR29b expression is lost in non-small cell lung cancer (NSCLC) cell lines and stimulation of &bgr;-catenin independent signaling, via Wnt7a expression, in NSCLC cell lines results in increased expression of hsa-miR29b. Surprisingly, we also identify specific regulation of hsa-miR29b by Wnt7a but not by Wnt3, a ligand for &bgr;-catenin-dependent signaling. Interestingly, knockdown of hsa-miR29b was enough to abrogate the tumor suppressive effects of Wnt7a/Frizzled9 signaling in NSCLC cells, suggesting that hsa-miR29b is an important mediator of &bgr;-catenin independent signaling. Finally, we show for the first time that hsa-miR29b plays an important role as a tumor suppressor in lung cancer by targeting murine double mutant 2 (MDM2), revealing novel nodes for Wnt7a/Frizzled9-mediated regulation of NSCLC cell proliferation.

Heterotrimeric G-Protein, Gα16, Is a Critical Downstream Effector of Non-Canonical Wnt Signaling and a Potent Inhibitor of Transformed Cell Growth in Non Small Cell Lung Cancer

G-protein-coupled receptors (GPCR) are the largest family of cell surface molecules that play important role/s in a number of biological and pathological processes including cancers. Earlier studies have highlighted the importance of Wnt7a signaling via its cognate receptor Frizzled9, a GPCR, in inhibition of cell proliferation, anchorage-independent growth, and reversal of transformed phenotype in non small cell lung cancer primarily through activation of the tumor suppressor, PPARγ. However, the G-protein effectors that couple to this important tumor suppressor pathway have not been identified, and are of potential therapeutic interest. In this study, by using two independent Wnt7a/Frizzled9-specific read-outs, we identify Gα16 as a novel downstream effector of Wnt7a/Frizzled9 signaling. Interestingly, Gα16 expression is severely down-regulated, both at the messenger RNA levels and protein levels, in many non small cell lung cancer cell lines. Additionally, through gene-specific knock-downs and expression of GTPase-deficient forms (Q212L) of Gα16, we also establish Gα16 as a novel regulator of non small cell lung cancer cell proliferation and anchorage-independent cell growth. Taken together, our data not only establish the importance of Gα16 as a critical downstream effector of the non-canonical Wnt signaling pathway but also as a potential therapeutic target for the treatment of non small cell lung cancer.

Erratum: Wnt7a is a novel inducer of β-catenin-independent tumor-suppressive cellular senescence in lung cancer (Oncogene (2015) 34 (5406) DOI:10.1038/onc.2015.165)

Correction to: Oncogene (2015) 34, 0000–0000; doi:10.1038/onc.2015.2; published online 2 March 2015 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.

K-homology splicing regulatory protein (KSRP) promotes post-transcriptional destabilization of Spry4 transcripts in non-small cell lung cancer

AU-rich element-binding proteins (ARE-BPs) offer post-transcriptional regulation of gene expression via physical interaction and recruitment of RNA decay machinery to the AU-rich elements within the 3′-UTR of the target transcripts. However, the role of ARE-BPs in lung cancer remains poorly understood. In this study, we have identified that K-homology splicing regulatory protein (KSRP), an ARE-BP, is robustly up-regulated in human lung cancer. Importantly, Kaplan-Meier survival analysis indicated that elevated KSRP expression was correlated with poor overall survival of lung cancer patients. Furthermore, cigarette smoke, a leading risk factor for lung cancer, was also identified to be an important contributor to increased KSRP expression. Remarkably, silencing of KSRP decreased cell proliferation, reversed anchorage-independent growth, and reduced migration/invasion, suggesting an oncogenic role for KSRP in lung cancer. Finally, we provide mechanistic evidence that KSRP promotes the down-regulation of Spry4 by a previously unidentified mechanism, i.e. post-transcriptional mRNA regulation.