Differential reprogramming of isogenic colorectal cancer cells by distinct activating KRAS mutations.

Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼ 16% of all cancer cases. Among the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumor type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS.

Conditional deletion of Stat3 in mammary epithelium impairs the acute phase response and modulates immune cell numbers during post-lactational regression.

Mammary gland regression following weaning (involution) is associated with extensive cell death and the acquisition of an inflammatory signature. Characterizing the interplay between mammary epithelial cells, the re-emerging stroma and immune cells has implications for the understanding of the pathogenesis of pregnancy-associated breast cancer. Stat3 has a role in orchestrating cell death and involution, and we sought to determine whether expression of Stat3 by the mammary epithelium also influences the innate immune environment and inflammatory cell influx in the gland. We examined mice in which Stat3 is conditionally deleted only in the mammary epithelium. Distinct sets of genes associated with the acute phase response and innate immunity are markedly up-regulated during first phase involution in a Stat3-dependent manner. During second phase involution, chitinase 3-like 1, which has been associated with wound healing and chronic inflammatory conditions, is dramatically up-regulated by Stat3. Also at this time, the number of mammary macrophages and mast cells increases per unit area, and this increase is impaired in the absence of epithelial Stat3. Furthermore, expression of arginase-1 and Ym1, markers of alternatively activated macrophages, is significantly decreased in the absence of Stat3, whilst iNOS, a marker associated with classically activated macrophages, shows significantly increased expression in the Stat3-deleted glands. Thus, Stat3 is a key transcriptional regulator of genes associated with innate immunity and wound healing and influences mammary macrophage and mast cell numbers. The presence of epithelial Stat3 appears to polarize the macrophages and epithelial cells towards an alternatively activated phenotype, since in the absence of Stat3, the gland retains a phenotype associated with classically activated macrophages. These findings have relevance to the study of pregnancy-associated breast cancer and the role of Stat3 signalling in recruitment of alternatively activated tumour-associated macrophages in breast cancer.

Key signalling nodes in mammary gland development and cancer. Signalling downstream of PI3 kinase in mammary epithelium: a play in 3 Akts.

The protein serine/threonine kinase Akt, also known as protein kinase B (PKB), is arguably the most important signalling nexus in the cell. Akt integrates a plethora of extracellular signals to generate diverse outcomes, including proliferation, motility, growth, glucose homeostasis, survival, and cell death. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is the second most frequently mutated pathway in cancer, after p53, and mutations in components of this pathway are found in around 70% of breast cancers. Thus, understanding how Akt relays input signals to downstream effectors is critically important for the design of therapeutic strategies to combat breast cancer. In this review, we will discuss the various signals upstream of Akt that impact on its activity, how Akt integrates these signals and modulates the activity of downstream targets to control mammary gland development, and how mutations in components of the pathway result in breast cancer.

ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL.

The proapoptotic protein Bim is expressed de novo following withdrawal of serum survival factors. Here, we show that Bim-/- fibroblasts and epithelial cells exhibit reduced cell death following serum withdrawal in comparison with their wild-type counterparts. In viable cells, Bax associates with Bcl-2, Bcl-x(L) and Mcl-1. Upon serum withdrawal, newly expressed Bim(EL) associates with Bcl-x(L) and Mcl-1, coinciding with the dissociation of Bax from these proteins. Survival factors can prevent association of Bim with pro-survival proteins by preventing Bim expression. However, we now show that even preformed Bim(EL)/Mcl-1 and Bim(EL)/Bcl-x(L) complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for Bim(EL) and requires ERK1/2-dependent phosphorylation of Bim(EL) at Ser(65). Finally, ERK1/2-dependent dissociation of Bim(EL) from Mcl-1 and Bcl-x(L) may play a role in regulating Bim(EL) degradation, since mutations in the Bim(EL) BH3 domain that disrupt binding to Mcl-1 cause increased turnover of Bim(EL). These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway.

Cigarette smoke prevents apoptosis through inhibition of caspase activation and induces necrosis.

Emphysema is characterized by enlargement of the distal airspaces in the lungs due to destruction of alveolar walls. Alveolar endothelial and epithelial cell apoptosis induced by cigarette smoke is thought to be a possible mechanism for this cell loss. In contrast, our studies show that cigarette smoke condensate (CSC) induces necrosis in alveolar epithelial cells and human umbilical vein endothelial cells. Furthermore, study of the cell death pathway in a model system using Jurkat cells revealed that in addition to inducing necrosis, CSC inhibited apoptosis induced by staurosporine or Fas ligation, with both effects prevented by the antioxidants glutathione and dithiothreitol. Time course experiments revealed that CSC inhibited an early step in the caspase cascade, whereby caspase-3 was not activated. Moreover, cell-free reconstitution of the apoptosome in cytoplasmic extracts from CSC-treated cells, by addition of cytochrome-c and dATP, did not result in activation of caspases-3 or -9. Thus, smoke treatment may alter the levels of pro- and antiapoptogenic factors downstream of the mitochondria to inhibit active apoptosome formation. Therefore, unlike previous studies, cell death in response to cigarette smoke by necrosis and not apoptosis may be responsible for the loss of alveolar walls and inflammation observed in emphysema.