Mechanisms linking mitogenic and growth inhibitory cytokine signaling and the cell cycle have not been fully elucidated in either cancer or in normal cells. Here we show that activation of protein kinase B (PKB)/Akt, contributes to resistance to antiproliferative signals and breast cancer progression in part by impairing the nuclear import and action of p27. Akt transfection caused cytoplasmic p27 accumulation and resistance to cytokine-mediated G1 arrest. The nuclear localization signal of p27 contains an Akt consensus site at threonine 157, and p27 phosphorylation by Akt impaired its nuclear import in vitro. Akt phosphorylated wild-type p27 but not p27T157A. In cells transfected with constitutively active Akt(T308DS473D)(PKB(DD)), p27WT mislocalized to the cytoplasm, but p27T157A was nuclear. In cells with activated Akt, p27WT failed to cause G1 arrest, while the antiproliferative effect of p27T157A was not impaired. Cytoplasmic p27 was seen in 41% (52 of 128) of primary human breast cancers in conjunction with Akt activation and was correlated with a poor patient prognosis. Thus, we show a novel mechanism whereby Akt impairs p27 function that is associated with an aggressive phenotype in human breast cancer.

Cell adhesion to substratum has been shown to regulate cyclin A expression as well as cyclin D-and E-dependent kinases, the latter via the up-regulation of cyclin D1 and the down-regulation of cyclin-Cdk inhibitors p21 and p27, respectively. This adhesion-dependent regulation of cell cycle is thought to be mediated by integrins. Here we demonstrate that stable transfection and overexpression of the integrin-linked kinase (ILK), which interacts with the ␤1 and ␤3 integrin cytoplasmic domains, induces anchorage-independent cell cycle progression but not serum-independent growth of rat intestinal epithelial cells (IEC18). ILK overexpression results in increased expression of cyclin D1, activation of Cdk4 and cyclin E-associated kinases, and hyperphosphorylation of the retinoblastoma protein. Normal, untransformed epithelial cells require anchorage to a substratum for cell growth and survival. Adhesion to the extracellular matrix (ECM) 1 is required for progression of cells through the G 1 and into the S phase of the cell cycle. When forced to remain in suspension, such cells arrest in the G 1 phase of the cell cycle and undergo apoptosis (1-3). Oncogenic transformation frequently induces anchorage-independent growth, in vitro, and is a specific correlate of tumor growth in vivo (4,5).In fibroblasts, cell adhesion has recently been demonstrated to regulate cell cycle progression by inducing the expression of cyclin D1 (6), the activation of cyclin E-Cdk2 (6, 7), and phosphorylation of the retinoblastoma protein (Rb) (6). Fibroblast adhesion also results in the down-regulation of expression of the Cdk inhibitor proteins, p21 and p27 (6, 7). The combined, adhesion-dependent elevation in cyclin D1 and the decrease in the expression of p21 and p27 result in the stimulation of cyclin D-Cdk4 and cyclin E-Cdk2 activities, both of which can phosphorylate Rb. This latter event relieves the restriction of the entry of cells into S phase, presumably by the release of the transcription factor E2F from phosphorylated Rb (8, 9). In some cell types the expression of cyclin A is also regulated in an anchorage-dependent manner (3, 10, 11), and anchorage-independent growth induced by activated Ras has been shown to depend on cyclin A expression (11). However, in these latter experiments cyclin D1 expression (12) and cyclin E-dependent kinase activity (11) were also dependent on Ras activation. Although mitogens can also activate cyclin D-and cyclin E-dependent kinases, cell adhesion per se can regulate these activities. The regulation of G 1 Cdks, therefore, requires the convergence of signals from both growth factors as well as from the ECM.Anchorage of cells to the ECM is mediated to a large extent by integrins, a large family of heterodimeric cell surface receptors (13, 14). The interaction of integrins with ECM ligands results in the transduction of intracellular signals leading to stimulation of tyrosine phosphorylation (15, 16), turnover of phosphoinositides (17), and activation of the Ras-mitogen-activated protein kinase (MA...

The successes of targeted drugs with companion predictive biomarkers and the technological advances in gene sequencing have generated enthusiasm for evaluating personalized cancer medicine strategies using genomic profiling. We assessed the feasibility of incorporating real-time analysis of somatic mutations within exons of 19 genes into patient management. Blood, tumor biopsy and archived tumor samples were collected from 50 patients recruited from four cancer centers. Samples were analyzed using three technologies: targeted exon sequencing using Pacific Biosciences PacBio RS, multiplex somatic mutation genotyping using Sequenom MassARRAY and Sanger sequencing. An expert panel reviewed results prior to reporting to clinicians. A clinical laboratory verified actionable mutations. Fifty patients were recruited. Nineteen actionable mutations were identified in 16 (32%) patients. Across technologies, results were in agreement in 100% of biopsy specimens and 95% of archival specimens. Profiling results from paired archival/biopsy specimens were concordant in 30/34 (88%) patients. We demonstrated that the use of next generation sequencing for real-time genomic profiling in advanced cancer patients is feasible. Additionally, actionable mutations identified in this study were relatively stable between archival and biopsy samples, implying that cancer mutations that are good predictors of drug response may remain constant across clinical stages.International efforts to quantify and catalogue mutations, gene expression and epigenetic data for multiple forms of cancer, coupled with the successes of targeted agents in patients with molecularly defined tumors and improvements in genomic technology, have increased enthusiasm to adopt genomic profiling into clinical cancer practice. 1 As the numbers of clinically significant genetic variants have increased, clinical testing has evolved, moving from single mutations to multiplex hotspot evaluations in multiple cancer genes. Several pilot studies have demonstrated the feasibility and potential benefits of real-time multiplex hotspot evaluations in various cancer types. 2-8 However, as improvements in genomic technology overcome previous concerns of cost, complexity, time and tissue requirements, an increasing interest in adopting next generation sequencing (NGS) for genomic profiling in clinical cancer practice has developed. 6,9,10 Roychowdhury et al. recently reported the use of integrative sequencing in the clinic and demonstrated its potential to facilitate biomarker driven clinical trials. 11 However, it remains unclear whether the use of high-throughput, real-time NGS for genomic profiling is capable of generating results in a timeframe that allows for changes to patient management. Furthermore, the additional value of NGS over the multiplex hotspot genotyping approach is unclear, and

The molecular mechanisms that regulate the synthesis of the myometrial gap junction protein, connexin-43 (Cx-43), are controversial. We measured myometrial Cx-43 messenger RNA, protein and gap junction frequency, and area in myometrial samples collected from nonpregnant rats and pregnant rats at days 5, 10, 15, 17, 18, 19, 20, 21, 22, 23 (during delivery), and 1 day postpartum and correlated these data with plasma concentrations of estradiol 17 beta and progesterone. Cx-43 transcripts were low or undetectable (connexin-43:glyceraldehyde phosphate dehydrogenase ratio < 0.2) in nonpregnant rats or in rats before day 10 of pregnancy. Transcripts rose to 0.52 +/- 0.11 on day 10, increased (2.9-fold) to 1.51 +/- 0.48 on day 22, and increased a further 2.9-fold to maximal levels of 4.42 +/- 0.67 during labor. Cx-43 protein was barely detectable on day 21 [0.12 +/- 0.04 relative optical density (ROD) units], increased 2.5-fold on day 22 (0.30 +/- 0.04 ROD units), and a further 3.7-fold during delivery (1.10 +/- 0.15 ROD units), at a time when gap junctions were present in large numbers in the cell membrane. Between day 21 and delivery the increase in Cx-43 transcripts (8.2-fold) and protein (9.2-fold) were of a similar magnitude. There was a significant positive correlation between the increases in Cx-43 transcripts and the increase in the ratio of plasma estradiol to progesterone. Levels of Cx-43 transcripts, protein, and gap junctions fell rapidly postpartum. Our data demonstrate: 1) that transcripts encoding the gap junction protein, Cx-43, are at maximal levels during delivery and that this increase is temporally associated with increases in Cx-43 protein and the appearance of gap junctions; and 2) that these data, in association with changes in plasma steroid concentrations, are consistent with myometrial Cx-43 transcript levels being regulated positively by estrogen and negatively by progesterone during pregnancy.