The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma is a poorly understood key event in breast tumor progression. Here, we analyzed the role of myoepithelial cells and fibroblasts in the progression of in situ carcinomas using a model of human DCIS and primary breast tumors. Progression to invasion was promoted by fibroblasts and inhibited by normal myoepithelial cells. Molecular profiles of isolated luminal epithelial and myoepithelial cells identified an intricate interaction network involving TGFbeta, Hedgehog, cell adhesion, and p63 required for myoepithelial cell differentiation, the elimination of which resulted in loss of myoepithelial cells and progression to invasion.

c When explanted into culture, normal human cells exhibit a finite number of cell divisions before entering a proliferative arrest termed replicative senescence. To identify genes essential for entry into replicative senescence, we performed an RNA interference (RNAi)-based loss-of-function screen and found that suppression of the Never in Mitosis Gene A (NIMA)-related protein kinase gene NEK4 disrupted timely entry into senescence. NEK4 suppression extended the number of population doublings required to reach replicative senescence in several human fibroblast strains and resulted in decreased transcription of the cyclin-dependent kinase inhibitor p21. NEK4-suppressed cells displayed impaired cell cycle arrest in response to double-stranded DNA damage, and mass spectrometric analysis of Nek4 immune complexes identified a complex containing DNA-dependent protein kinase catalytic subunit [DNA-PK(cs)], Ku70, and Ku80. NEK4 suppression causes defects in the recruitment of DNA-PK(cs) to DNA upon induction of double-stranded DNA damage, resulting in reduced p53 activation and H2AX phosphorylation. Together, these observations implicate Nek4 as a novel regulator of replicative senescence and the response to double-stranded DNA damage. W hen explanted into culture, human cells derived from normal tissues exhibit a finite number of cell divisions (29).After extended passage in culture, human cells begin to divide more slowly and eventually enter an irreversible proliferative arrest termed replicative senescence (58). Senescent cells exhibit a characteristic large, flattened morphology and, although metabolically active, have permanently exited the cell cycle. Since senescence prevents cells from exceeding a defined replicative limit, replicative senescence has been implicated in aging, as well as in tumor suppression. In support of this hypothesis, several studies have identified senescent cells in premalignant lesions (8,(14)(15)(16)41), and genetic mutations commonly found in human cancers, such as loss of the TP53 and RB1 tumor suppressors or constitutive expression of telomerase, have also been shown to interfere with replicative senescence (26,32).Oncogenic stress, DNA damage, replication fork stalling, acute telomere uncapping, and other types of cellular stress can induce an acute senescence response with hallmarks similar to those seen in cells that have entered replicative senescence (37, 39, 50). Several lines of evidence implicate a common set of pathways in the regulation of acute and replicative senescence, although the extent of overlapping pathways is not clear. The expression of the simian virus 40 (SV40) oncoprotein large T antigen (LT) permits human cells to bypass replicative senescence (51). LT binds and inactivates p53 and RB, resulting in a loss of cell cycle checkpoints required to enforce the senescent state. However, in some cell types, senescence is dependent solely upon the tumor suppressor p53, and inactivation of p53 alone is sufficient to bypass replicative senescence or to extend the time t...

POT1 is a 3 ¶ telomeric single-stranded overhang binding protein that has been implicated in chromosome end protection, the regulation of telomerase function, and defining the 5 ¶ chromosome terminus. In human cancer cells that exhibit constitutive hTERT activity, hPOT1 exerts control over telomere length. Primary human fibroblasts express low levels of catalytically active hTERT in an S-phase -restricted manner that fails to counteract telomere attrition with cell division. Here, we show that diploid human fibroblasts in which hPOT1 expression has been suppressed harbor telomeres that are longer than control cells. This difference in telomere length delays the onset of replicative senescence and is dependent on S-phase -restricted hTERT expression. These findings are consistent with the view that hPOT1 promotes a nonextendable telomere state resistant to extension by S-phase -restricted telomerase. Manipulating this function of hPOT1 may thus hasten the cytotoxic effects of telomerase inhibition. (Mol Cancer Res 2008;6(10):1582 -93)