Increased alpha-actinin-1 destabilizes E-cadherin-based adhesions and associates with poor prognosis in basal-like breast cancer

The controlled formation and stabilization of E-cadherin-based adhesions is vital for epithelial integrity. This requires co-operation between the E-cadherin-based adhesions and the associated actin cytoskeleton. In cancer, this co-operation often fails, predisposing cells to migration through molecular mechanisms that have only been partially characterized. Here, we demonstrate that the actin filament cross-linker alpha-actinin-1 is frequently increased in human breast cancer. In mammary epithelial cells, the increased alpha-actinin-1 levels promote cell migration and induce disorganized acini-like structures in Matrigel. This is accompanied by a major reorganization of the actin cytoskeleton and the associated E-cadherin-based adhesions. Increased expression of alpha-actinin-1 is particularly noted in basal-like breast cancer cell lines, and in breast cancer patients it associates with poor prognosis in basal-like subtypes. Downregulation of alpha-actinin-1 in E-cadherin expressing basal-like breast cancer cells demonstrate that alpha-actinin-1-assembled actin fibers destabilize E-cadherin-based adhesions. Taken together, these results indicate that increased alpha-actinin-1 expression destabilizes E-cadherin-based adhesions, which is likely to promote the migratory potential of breast cancer cells. Furthermore, our results identify alpha-actinin-1 as a candidate prognostic biomarker in basal-like breast cancer.Peer reviewe

Depletion of Mediator Kinase Module Subunits Represses Superenhancer-Associated Genes in Colon Cancer Cells

In cancer, oncogene activation is partly mediated by acquired superenhancers, which therefore represent potential targets for inhibition. Superenhancers are enriched for BRD4 and Mediator, and both BRD4 and the Mediator MED12 subunit are disproportionally required for expression of superenhancer-associated genes in stem cells. Here we show that depletion of Mediator kinase module subunit MED12 or MED13 together with MED13L can be used to reduce expression of cancer-acquired superenhancer genes, such as the MYC gene, in colon cancer cells, with a concomitant decrease in proliferation. Whereas depletion of MED12 or MED13/MED13L caused a disproportional decrease of superenhancer gene expression, this was not seen with depletion of the kinases cyclin-dependent kinase 9 (CDK8) and CDK19. MED12-MED13/MED13L-dependent superenhancer genes were coregulated by beta-catenin, which has previously been shown to associate with MED12. Importantly, beta-catenin depletion caused reduced binding of MED12 at the MYC superenhancer. The effect of MED12 or MED13/MED13L depletion on cancer-acquired superenhancer gene expression was more specific than and partially distinct from that of BRD4 depletion, with the most efficient inhibition seen with combined targeting. These results identify a requirement of MED12 and MED13/MED13L for expression of acquired superenhancer genes in colon cancer, implicating these Mediator subunits as potential therapeutic targets for colon cancer, alone or together with BRD4.Peer reviewe

Regulation of the Mcs2 C-type cyclin in fission yeast

CDK activation requires activating phosphorylation of a conserved residue in the T-loop by the CAK (CDK-activating kinase). In both fission yeast and higher eukaryotes, CAK is a trimeric complex composed of Mcs6-Mcs2-Pmh1 or its homologue [1–3]. Two hybrid assays revealed interaction of both Mcs2 and Pmh1 with Shp1, a subunit of the SCF ubiquitin ligase. This prompted us to test the stability of these CAK regulators. Although the steady-state level of Mcs2 does not seem to change during the cell cycle [4], we show that it is strongly correlated to Mcs6 kinase activity and that Mcs2 was nearly undetectable when Mcs6 was strongly overexpressed. Interestingly, this effect is reversed by a mutation in shp1. Taken together, these results suggest a putative regulation of Mcs2 by Mcs6 phosphorylation and SCF ubiquitin ligase complex. Thus, the hypothesis is under investigation. (1) Hermand D, Westerling T. Pihlak A, et al. 2001. EMBO J 20: 82–90. (2) Hermand D, Pihlak A, Westerling T, et al. 1998. EMBO J 17: 7230–7238. (3) Kaldis P. 1999. Cell Mol Life Sci 55: 284–296. (4) Molz L, Beach D. 1993. EMBO J 12: 1723–1732.S.B. is a FNRS Research Fellow. D.H. is a FNRS Scientific Research Worker. L.T. is recipient of a FRIA Fellowship.info:eu-repo/semantics/publishe

Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast

Here we report functional characterization of the essential fission yeast Skp1 homologue. We have created a conditional allele of skp1 (skp1-3f) mimicking the mutation in the budding yeast skp1-3 allele. Although budding yeast skp1-3 arrests at the G(1)/S transition, skp1-3f cells progress through S phase and instead display two distinct phenotypes. A fraction of the skp1-3f cells arrest in mitosis with high Cdc2 activity. Other skp1-3f cells as well as the skp1-deleted cells accumulate abnormal thick septa leading to defects in cell separation. Subsequent identification of 16 fission yeast F-box proteins led to identification of the product of pof6 (for pombe F-box) as a Skp1-associated protein. Interestingly, cells deleted for the essential pof6 gene display a similar cell separation defect noted in skp1 mutants, and Pof6 localizes to septa and cell tips. Purification of Pof6 demonstrates association of Skp1, whereas the Pcu1 cullin was absent from the complex. These findings reveal an essential non-Skp1-Cdc53/Cullin-F-box protein function for the fission yeast Skp1 homologue and the F-box protein Pof6 in cell separation.info:eu-repo/semantics/publishe