Signaling Networks Associated with AKT Activation in Non-Small Cell Lung Cancer (NSCLC): New Insights on the Role of Phosphatydil-Inositol-3 kinase

Aberrant activation of PI3K/AKT signalling represents one of the most common molecular alterations in lung cancer, though the relative contribution of the single components of the cascade to the NSCLC development is still poorly defined. In this manuscript we have investigated the relationship between expression and genetic alterations of the components of the PI3K/AKT pathway [KRAS, the catalytic subunit of PI3K (p110α), PTEN, AKT1 and AKT2] and the activation of AKT in 107 surgically resected NSCLCs and have analyzed the existing relationships with clinico-pathologic features. Expression analysis was performed by immunohistochemistry on Tissue Micro Arrays (TMA); mutation analysis was performed by DNA sequencing; copy number variation was determined by FISH. We report that activation of PI3K/AKT pathway in Italian NSCLC patients is associated with high grade (G3–G4 compared with G1–G2; n = 83; p<0.05) and more advanced disease (TNM stage III vs. stages I and II; n = 26; p<0.05). In addition, we found that PTEN loss (41/104, 39%) and the overexpression of p110α (27/92, 29%) represent the most frequent aberration observed in NSCLCs. Less frequent molecular lesions comprised the overexpression of AKT2 (18/83, 22%) or AKT1 (17/96, 18%), and KRAS mutation (7/63, 11%). Our results indicate that, among all genes, only p110α overexpression was significantly associated to AKT activation in NSCLCs (p = 0.02). Manipulation of p110α expression in lung cancer cells carrying an active PI3K allele (NCI-H460) efficiently reduced proliferation of NSCLC cells in vitro and tumour growth in vivo. Finally, RNA profiling of lung epithelial cells (BEAS-2B) expressing a mutant allele of PIK3 (E545K) identified a network of transcription factors such as MYC, FOS and HMGA1, not previously recognised to be associated with aberrant PI3K signalling in lung cancer

Low-sodium diet induces atherogenesis regardless of lowering blood pressure in hypertensive hyperlipidemic mice.

This study investigated the influence of sodium restriction and antihypertensive drugs on atherogenesis utilizing hypertensive (H) low-density lipoprotein-receptor knockout mice treated or not with losartan (Los) or hydralazine (Hyd) and fed low-sodium (LS) or normal-sodium (NS) chow. Despite reducing the blood pressure (BP) of H-LS mice, the LS diet caused arterial lipid infiltration due to increased plasma total cholesterol (TC) and triglycerides (TG). Los and Hyd reduced the BP of H-LS mice, and Los effectively prevented arterial injury, likely by reducing plasma TG and nonesterified fatty acids. Aortic lipid infiltration was lower in Los-treated H-LS mice (H-LS+Los) than in normotensive (N)-LS and H-LS mice. Aortic angiotensin II type 1 (AT1) receptor content was greater in H-NS than H-LS mice and in H-LS+Hyd than H-LS+Los mice. Carboxymethyl-lysine (CML) and receptor for advanced glycation end products (RAGE) immunostaining was greater in H-LS than H-NS mice. CML and RAGE levels were lower in LS animals treated with antihypertensive drugs, and Hyd enhanced the AT1 receptor level. Hyd also increased the gene expression of F4/80 but not tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-10, intercellular adhesion molecule-1 or cluster of differentiation 66. The novelty of the current study is that in a murine model of simultaneous hypertension and hyperlipidemia, the pleiotropic effect of chronic, severe sodium restriction elicited aortic damage even with reduced BP. These negative effects on the arterial wall were reduced by AT1 receptor antagonism, demonstrating the influence of angiotensin II in atherogenesis induced by a severely LS diet

Low-sodium diet induces atherogenesis regardless of lowering blood pressure in hypertensive hyperlipidemic mice - Fig 5

<p>Histomorphometric analysis of immunofluorescence-stained AT1 receptor (<b>A</b> segments I and II; <b>B</b> segments III and IV), and vascular injury quantified by a histomorphometric analysis of immunofluorescence-stained CML (<b>C</b> segments I and II; <b>D</b> segments III and IV) and RAGE (<b>E</b> segments I and II; <b>F</b> segments III and IV); data are represented as the mean percentage of the total positively stained area of the aortic arch cross-sections; n = 4 mice per group. ^a <i>P</i> < 0.05, hypertensive mice fed a normal-sodium diet (H-NS) <i>vs</i> hypertensive mice fed a low-sodium diet (H-LS), Mann Whitney test. ^b <i>P</i> < 0.05, Kruskal Wallis with Dunn’s post hoc test applied for comparisons among LS groups.</p

Representative examples of lipid infiltration visualized by oil red O staining of the intima and media layers of different aortic arch segments from animal groups consisting of hypertensive mice fed a normal-sodium diet (H-NS), normotensive mice fed a low-sodium diet (N-LS), hypertensive mice fed a low-sodium diet (H-LS), hypertensive mice fed a low-sodium diet and treated with losartan (H-LS+Los) and hypertensive mice fed a low-sodium diet and treated with hydralazine (H-LS+Hyd).

<p>Arrows indicate sites of aortic arch lipid infiltration. ADV = adventitia, MED = media, INT = intima (400×).</p

Gene expression (mRNA) of the AT1 receptor (<i>Agtr1</i>) and RAGE (<i>Ager</i>) in the mouse aortic arch.

<p>Data are expressed as relative mRNA units normalized to mouse β2M expression. Mann Whitney test was used for comparisons between hypertensive mice fed a normal-sodium (H-NS) diet and hypertensive mice fed a low-sodium (H-LS) diet. The Kruskal Wallis test with Dunn’s post hoc test was applied for comparisons among the LS groups; n ≥ 4 mice per group.</p

Gene-specific RT-PCR primers.

<p>Gene-specific RT-PCR primers.</p

Univariate regression analysis of arterial lipid infiltration in N and H LDLR KO mice fed a NS or LS diet treated or not with an antihypertensive drug.

<p>Univariate regression analysis of arterial lipid infiltration in N and H LDLR KO mice fed a NS or LS diet treated or not with an antihypertensive drug.</p

Phenotypic characteristics of normotensive (N) or hypertensive (H) LDLR KO mice fed either a normal-sodium (NS) or a low-sodium (LS) diet and treated or not with losartan (Los) or hydralazine (Hyd).

<p>Phenotypic characteristics of normotensive (N) or hypertensive (H) LDLR KO mice fed either a normal-sodium (NS) or a low-sodium (LS) diet and treated or not with losartan (Los) or hydralazine (Hyd).</p