Nedd9 Restrains Autophagy to Limit Growth of Early Stage Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most common cancer worldwide. With overall 5-year survival estimated at <17%, it is critical to identify factors that regulate NSCLC disease prognosis. NSCLC is commonly driven by mutations in KRAS and TP53, with activation of additional kinases such as SRC promoting tumor invasion. In this study, we investigated the role of NEDD9, a SRC activator and scaffolding protein, in NSCLC tumorigenesis. In an inducible model of NSCLC dependent on Kras mutation and Trp53 loss (KP mice), deletion of Nedd9 (KPN mice) led to the emergence of larger tumors characterized by accelerated rates of tumor growth and elevated proliferation. Orthotopic injection of KP and KPN tumors into the lungs of Nedd9-wild-type and -null mice indicated the effect of Nedd9 loss was cell-autonomous. Tumors in KPN mice displayed reduced activation of SRC and AKT, indicating that activation of these pathways did not mediate enhanced growth of KPN tumors. NSCLC tumor growth has been shown to require active autophagy, a process dependent on activation of the kinases LKB1 and AMPK. KPN tumors contained high levels of active LKB1 and AMPK and increased autophagy compared with KP tumors. Treatment with the autophagy inhibitor chloroquine completely eliminated the growth advantage of KPN tumors. These data for the first time identify NEDD9 as a negative regulator of LKB1/AMPK-dependent autophagy during early NSCLC tumor growth. SIGNIFICANCE: This study demonstrates a novel role for the scaffolding protein NEDD9 in regulating LKB1-AMPK signaling in early stage non-small cell lung cancer, suppressing autophagy and tumor growth.

Fibroblast growth factor 2 causes G2/M cell cycle arrest in ras-driven tumor cells through a Src-dependent pathway.

We recently reported that paracrine Fibroblast Growth Factor 2 (FGF2) triggers senescence in Ras-driven Y1 and 3T3(Ras) mouse malignant cell lines. Here, we show that although FGF2 activates mitogenic pathways in these Ras-dependent malignant cells, it can block cell proliferation and cause a G2/M arrest. These cytostatic effects of FGF2 are inhibited by PD173074, an FGF receptor (FGFR) inhibitor. To determine which downstream pathways are induced by FGF2, we tested specific inhibitors targeting mitogen-activated protein kinase (MEK), phosphatidylinositol 3 kinase (PI3K) and protein kinase C (PKC). We show that these classical mitogenic pathways do not mediate the cytostatic activity of FGF2. On the other hand, the inhibition of Src family kinases rescued Ras-dependent malignant cells from the G2/M irreversible arrest induced by FGF2. Taken together, these data indicate a growth factor-sensitive point in G2/M that likely involves FGFR/Ras/Src pathway activation in a MEK, PI3K and PKC independent manner.

Dietary supplementation of lutein reduces colon carcinogenesis in DMH-treated rats by modulating K-ras, PKB, and ß-catenin proteins.

In colon cancer, disturbances have been detected in genes coding for proteins involved in cellular proliferation, such as K-ras, ß-catenin, extracellular signal-regulated kinases (ERKs), and the protein kinase B (PKB). Although carotenoids such as lutein have an important role to prevent and treat some types of cancer, there are very few studies about the effect of lutein against colon cancer and its activity at the molecular level. Therefore, the aim of this study was to evaluate the chemoprotective activity of lutein against colon cancer induced by dimethylhydrazine (DMH). The results showed a significant increase in protein expression for K-ras and ß-catenin in tumors of DMH-treated rats. Simultaneously, we detected changes in the phosphorylation state of ERK1/2 and PKB in DMH-treated animals. Lutein given in the diet (0.002%), before (prevention) and after (treatment) DMH administration, diminished the number of tumors by 55% and 32%, respectively. Moreover, lutein significantly decreased in tumors the expression of K-ras (25%) and ß-catenin (28%) and the amount of pPKB (32%), during the prevention, and 39%, 26%, and 26% during the treatment stage, respectively. This study demonstrates the chemoprotective effect of lutein against colon cancer by modulating the proliferative activity of K-ras, PKB, and ß-catenin proteins.

Chromosomal rearrangements involving telomeric DNA sequences in Balb/3T3 cells transfected with the Ha-ras oncogene.

Chromosomal instability involving telomeric DNA sequences was studied in mouse Balb/3T3 fibroblasts transfected with a mutated human c-Ha-ras-1 gene (B61 cells) and spontaneously immortalized normal parental cells (A31 cells), using fluorescence in situ hybridization (FISH). FISH analysis with a telomeric probe revealed high frequencies of chromosome alterations involving telomeric regions, mainly stable and unstable Robertsonian fusion-like configurations (RLC) (0.25 and 1.95/cell in A31 and B61 cells, respectively) and chromosome ends lacking telomeric signals in one (LTS') or both chromatids (LTS") (5.9 and 17.5/cell for A31 and B61 cells, respectively). Interstitial telomeric sequences (ITS) were also detected at both non-telomeric sites and in the centromeres of RLC. The frequencies of RLCs with ITS located in the centromeres were 3-fold higher in B61 compared with A31 cells. We demonstrated a high level of chromosome instability involving telomeric DNA sequences in ras-transfected cells overexpressing ras mRNA, which could be a consequence of rapid cell cycle progression associated with a deficient telomere capping mechanism.

ACTH inhibits A Ras-dependent anti-apoptotic and mitogenic pathway in mouse Y1 adrenocortical cells.

Mouse Y1 adrenocortical tumor cells harbor amplified and overexpressed c-Ki-ras gene, displaying relatively high constitutive levels of Ras x GTP. Here we report that Y1 cells also constitutively display high levels of phosphorylated AKT/PKB, that are dependent on Ras x GTP and PI3K. ACTH rapidly causes dephosphorylation of AKT/PKB in a cAMP/PKA dependent maner. This ACTH inhibition of the anti-apoptic and mitogenic AKT/PKB pathway is likely to be relevant in ACTH growth inhibitory effects in Y-adrenocortical cells.

Ha-ras oncogene transformation abolishes retinoic acid-induced reduction of intracellular fibronectin.

All-trans-retinoic acid (RA) is a master regulator of cell differentiation and in this process it greatly influences cell adhesion and the elaboration of the extracellular matrix. Therefore, we were interested in the effect of RA on the biosynthesis of fibronectin (FN). RA reduced the level of intracellular FN in a time- and concentration-dependent fashion in NIH-3T3 cells, but not in NIH-3T3 cells transformed by an activated Ha-ras oncogene. Since the steady-state level of FN transcripts did not change after treatment of the cells with RA for various times or concentrations, RA probably acts at the translational level. In NIH-3T3 cells, RA had distinct effects on different receptors, from decreasing retinoic acid receptor (RAR)alpha to increasing RAR beta expression to no effect on RAR gamma. Transformation of NIH-3T3 cells with an activated Ha-ras oncogene downmodulated RAR expression and also abolished responsiveness to RA. A variety of approaches permitted the following conclusions: 1) RA-dependent FN downmodulation is mediated by RARs, 2) retinoid X receptors (RXRs) mediate the observed reduction of RAR alpha by RA, and 3) the blockade of RA responsiveness by Ha-ras-transfected cells cannot be overcome by overexpression of RAR alpha. These studies have identified fibronectin and RAR alpha as RA targets in fibroblast cells and have shown that oncogenic transformation renders the cells resistant to RA action.