Metastasis is the major cause of carcinoma-induced death, but mechanisms involved are poorly understood. Metastasis crucially involves epithelial-to-mesenchymal transition (EMT), causing loss of epithelial polarity. Here we identify Annexin A1 (AnxA1), a protein with important functions in intracellular vesicle trafficking, as an efficient suppressor of EMT and metastasis in breast cancer. AnxA1 levels were strongly reduced in EMT of mammary epithelial cells, in metastatic murine and human cell lines and in metastatic mouse and human carcinomas. RNAi-mediated AnxA1 knockdown cooperated with oncogenic Ras to induce TGFβ-independent EMT and metastasis in non-metastatic cells. Strikingly, forced AnxA1 expression in metastatic mouse and human mammary carcinoma cells reversed EMT and abolished metastasis. AnxA1 knockdown stimulated multiple signalling pathways but only Tyk2/Stat3 and Erk1/2 signalling were essential for EMT.

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Class switch recombination (CSR) at the immunoglobulin heavy chain (IgH) locus generates antibody isotypes. CSR depends on double-strand breaks (DSBs) induced by activation-induced cytidine deaminase (AID). Although DSB formation and repair machineries are active in G1 phase, efficient CSR is dependent on cell proliferation and S phase entry; however, the underlying mechanisms are obscure. Here, we show that efficient CSR requires the replicative helicase, the Mcm complex. Mcm proteins are enriched at IgH switch regions during CSR, leading to assembly of facultative replication origins that require Mcm helicase function for productive CSR. Assembly of CSR-associated origins is facilitated by R loops and promotes the physical proximity (synapsis) of recombining switch regions, which is reduced by R loop inhibition or Mcm complex depletion. Thus, R loops contribute to replication origin specification that promotes DSB resolution in CSR. This suggests a mechanism for the dependence of CSR on S phase and cell division.

Somatic hypermutation (SHM) of immunoglobulin variable regions in B cells modulates antibody-antigen affinity and is indispensable for adaptive immunity. Mutations are introduced by activation-induced cytidine deaminase (AID) in a co-transcriptional manner resulting in discrete mutation spectra. Current models propose that activating epigenetic marks, transcriptional pausing and convergent transcription are necessary for optimal AID recruitment. However, whether these or other transcriptional features can explain the discrete mutation spectra is unknown. To address this, we compared mutation and nascent transcription at single nucleotide resolution. Surprisingly, with this precision, SHM spectra do not correlate with any transcriptional feature at human and mouse variable regions and non-immunoglobulin AID targets. Moreover, SHM is resistant to up to four-fold reduction of both activating epigenetic marks and transcription. We propose that, following AID recruitment to its target genes, the DNA sequence flanking an AID target motif is the key determinant of mutability rather than the local transcriptional and chromatin landscape.