We analysed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, messenger RNA arrays, microRNA sequencing and reverse-phase protein arrays. Our ability to integrate information across platforms provided key insights into previously defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity. Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at >10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the luminal A subtype. We identified two novel protein-expression-defined subgroups, possibly produced by stromal/microenvironmental elements, and integrated analyses identified specific signalling pathways dominant in each molecular subtype including a HER2/phosphorylated HER2/EGFR/phosphorylated EGFR signature within the HER2-enriched expression subtype. Comparison of basal-like breast tumours with high-grade serous ovarian tumours showed many molecular commonalities, indicating a related aetiology and similar therapeutic opportunities. The biological finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biological subtypes of breast cancer.

/pdf/comprehensive-molecular-portraits-of-human-breast-tumours-4t6h37c9ow.pdf

Comprehensive molecular portraits of human breast tumours

Eukaryotic cells replicate by a complex series of evolutionarily conserved events that are tightly regulated at defined stages of the cell division cycle. Progression through this cycle involves a large number of dedicated protein complexes and signaling pathways, and deregulation of this process is implicated in tumorigenesis. We applied high-resolution mass spectrometry-based proteomics to investigate the proteome and phosphoproteome of the human cell cycle on a global scale and quantified 6027 proteins and 20,443 unique phosphorylation sites and their dynamics. Co-regulated proteins and phosphorylation sites were grouped according to their cell cycle kinetics and compared to publicly available messenger RNA microarray data. Most detected phosphorylation sites and more than 20% of all quantified proteins showed substantial regulation, mainly in mitotic cells. Kinase-motif analysis revealed global activation during S phase of the DNA damage response network, which was mediated by phosphorylation by ATM or ATR or DNA-dependent protein kinases. We determined site-specific stoichiometry of more than 5000 sites and found that most of the up-regulated sites phosphorylated by cyclin-dependent kinase 1 (CDK1) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells.

Quantitative Phosphoproteomics Reveals Widespread Full Phosphorylation Site Occupancy During Mitosis

Oscillations in the activity of cyclin-dependent kinases (CDKs) promote progression through the eukaryotic cell cycle. This review examines how proteolysis regulates CDK activity—by degrading CDK activators or inhibitors—and also how proteolysis may directly trigger the transition from metaphase to anaphase. Proteolysis during the cell cycle is mediated by two distinct ubiquitin-conjugation pathways. One pathway, requiring CDC34, initiates DNA replication by degrading a CDK inhibitor. The second pathway, involving a large protein complex called the anaphase-promoting complex or cyclosome, initiates chromosome segregation and exit from mitosis by degrading anaphase inhibitors and mitotic cyclins. Proteolysis therefore drives cell cycle progression not only by regulating CDK activity, but by directly influencing chromosome and spindle dynamics.

https://science.sciencemag.org/content/sci/274/5293/1652.full.pdf

How proteolysis drives the cell cycle

The anaphase promoting complex/cyclosome (APC/C) is a ubiquitin ligase that has essential functions in and outside the eukaryotic cell cycle. It is the most complex molecular machine that is known to catalyse ubiquitylation reactions, and it contains more than a dozen subunits that assemble into a large 1.5-MDa complex. Recent discoveries have revealed an unexpected multitude of mechanisms that control APC/C activity, and have provided a first insight into how this unusual ubiquitin ligase recognizes its substrates.

/pdf/the-anaphase-promoting-complex-cyclosome-a-machine-designed-4j8aewxc8c.pdf

The anaphase promoting complex/cyclosome: a machine designed to destroy

F-Box Proteins Are Receptors that Recruit Phosphorylated Substrates to the SCF Ubiquitin-Ligase Complex

Kinase-Selective Enrichment Enables Quantitative Phosphoproteomics of the Kinome across the Cell Cycle

We have established a one-hybrid screen that allows the in vivo localization of proteins at a functional Saccharomyces cerevisiae centromere. Applying this screen we have identified three proteins—Ctf19, Mcm21, and the product of an unspecified open reading frame that we named Okp1—as components of the budding yeast centromere. Ctf19, Mcm21, and Okp1 most likely form a protein complex that links CBF3, a protein complex directly associated with the CDE III element of the centromere DNA, with further components of the budding yeast centromere, Cbf1, Mif2, and Cse4. We demonstrate that the CDE III element is essential and sufficient to localize the established protein network to the centromere and propose that the interaction of the CDE II element with the CDE III localized protein complex facilitates a protein‐DNA conformation that evokes the active centromere.

/pdf/a-putative-protein-complex-consisting-of-ctf19-mcm21-and-wzmd6x2vkg.pdf

A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore.

Summary tion of sister chromatids and homologs in somatic andmeiotic cells, respectively, demonstrating that separase Stable maintenance of genetic information requires activity might be controlled by Cdk1 in vivo (Chang et chromosome segregation to occur with high accu- al., 2003; Hagting et al., 2002; Herbert et al., 2003). racy. Anaphase is triggered when ring-shaped cohesin We show here that securin-independent inhibition of is cleaved by separase, a protease regulated by asso- separase is a two-step mechanism consisting of (1) ciation with its inhibitor securin. Dispensability of ver- phosphorylation of separase and (2) stable binding of tebrate securin strongly suggests additional means Cdk1 to the protease. Unexpectedly, we find that verte- of separase regulation. Indeed, sister chromatid sepa- brate separase is a Cdk1 inhibitor that resembles yeast ration but not securin degradation is inhibited by con- Cdc6 in terms of Cdk1 binding. Securin and Cdk1 bind stitutively active cyclin-dependent kinase 1 (Cdk1)

Short Article Mutual Inhibition of Separase and Cdk1 by Two-Step Complex Formation

https://www.cell.com/molecular-cell/pdf/S1097-2765(05)01346-8.pdf

Mutual Inhibition of Separase and Cdk1 by Two-Step Complex Formation

While separase is implicated in centrosome disjunction as well as in chromosome separation, its precise role at the centrosome has been uncertain. The proteolytic activity of separase is demonstrated to induce centriole disengagement through cleavage of cohesin.

Olaf Stemmann

Papers

Kinase-Selective Enrichment Enables Quantitative Phosphoproteomics of the Kinome across the Cell Cycle

A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore.

Short Article Mutual Inhibition of Separase and Cdk1 by Two-Step Complex Formation

Mutual Inhibition of Separase and Cdk1 by Two-Step Complex Formation

Cleavage of cohesin rings coordinates the separation of centrioles and chromatids