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High-grade serous ovarian cancers (HGSOC) represent the most common subtype of ovarian malignancies. Due to the frequency of late-stage diagnosis and high rates of recurrence following standard of care treatments, novel therapies are needed to promote durable responses. We investigated the anti-tumor activity of CD3 T cell engaging bispecific antibodies (TCBs) directed against the PAX8 lineage-driven HGSOC tumor antigen LYPD1 and demonstrated that anti-LYPD1 TCBs induce T cell activation and promote in vivo tumor growth inhibition in LYPD1-expressing HGSOC. To selectively target LYPD1-expressing tumor cells with high expression while sparing cells with low expression, we coupled bivalent low-affinity anti-LYPD1 antigen-binding fragments (Fabs) with the anti-CD3 scFv. In contrast to the monovalent anti-LYPD1 high-affinity TCB (VHP354), the bivalent low-affinity anti-LYPD1 TCB (QZC131) demonstrated antigen density-dependent selectivity and showed tolerability in cynomolgus monkeys at the maximum dose tested of 3 mg/kg. Collectively, these data demonstrate that bivalent TCBs directed against LYPD1 have compelling efficacy and safety profiles to support its use as a treatment for high-grade serous ovarian cancers.

Metastasis: A matter of translation? Solid tumors shed a small number of cancer cells into the bloodstream, some of which are believed to contribute to metastasis. The molecular features that confer these circulating tumor cells (CTCs) with metastatic potential are poorly understood. Ebright et al. studied CTCs from breast cancer patients and found that cells with increased expression levels of certain ribosomal proteins and regulators of translation had greater metastatic capacity in a mouse model (see the Perspective by Ma and Jeffrey). Consistent with this finding, patients with higher levels of this subset of CTCs tended to have a poorer prognosis. Science , this issue p. 1468 ; see also p. 1424

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

Metastasis-competent circulating tumour cells (CTCs) experience oxidative stress in the bloodstream, but their survival mechanisms are not well defined. Here, comparing single-cell RNA-Seq profiles of CTCs from breast, prostate and lung cancers, we observe consistent induction of β-globin (HBB), but not its partner α-globin (HBA). The tumour-specific origin of HBB is confirmed by sequence polymorphisms within human xenograft-derived CTCs in mouse models. Increased intracellular reactive oxygen species (ROS) in cultured breast CTCs triggers HBB induction, mediated through the transcriptional regulator KLF4. Depletion of HBB in CTC-derived cultures has minimal effects on primary tumour growth, but it greatly increases apoptosis following ROS exposure, and dramatically reduces CTC-derived lung metastases. These effects are reversed by the anti-oxidant N-Acetyl Cysteine. Conversely, overexpression of HBB is sufficient to suppress intracellular ROS within CTCs. Altogether, these observations suggest that β-globin is selectively deregulated in cancer cells, mediating a cytoprotective effect during blood-borne metastasis., Circulating tumour cells contribute to metastatic spread. Here the authors find that beta-chain of haemoglobin is overexpressed in those cells and protects them from oxidative stress, prolonging their survival in circulation and thereby increasing the likelihood of metastasis formation.

SUMMARY Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

SummaryKinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

SummaryEpithelial stem cells self-renew while maintaining multipotency, but the dependence of stem cell properties on maintenance of the epithelial phenotype is unclear. We previously showed that trophoblast stem (TS) cells lacking the protein kinase MAP3K4 maintain properties of both stemness and epithelial-mesenchymal transition (EMT). Here, we show that MAP3K4 controls the activity of the histone acetyltransferase CBP, and that acetylation of histones H2A and H2B by CBP is required to maintain the epithelial phenotype. Combined loss of MAP3K4/CBP activity represses expression of epithelial genes and causes TS cells to undergo EMT while maintaining their self-renewal and multipotency properties. The expression profile of MAP3K4-deficient TS cells defines an H2B acetylation-regulated gene signature that closely overlaps with that of human breast cancer cells. Taken together, our data define an epigenetic switch that maintains the epithelial phenotype in TS cells and reveals previously unrecognized genes potentially contributing to breast cancer.

Epithelial-mesenchymal transition (EMT) is an essential developmental program that becomes reactivated in adult tissues to promote the progression of cancer. EMT has been largely studied by examining the beginning epithelial state or the ending mesenchymal state without studying the intermediate stages. Recent studies using trophoblast stem (TS) cells paused in EMT have defined the molecular and epigenetic mechanisms responsible for modulating the intermediate "metastable" stages of EMT. Targeted inactivation of MAP3K4, knockdown of CBP, or overexpression of SNAI1 in TS cells induced similar metastable phenotypes. These TS cells exhibited epigenetic changes in the histone acetylation landscape that cause loss of epithelial maintenance while preserving self-renewal and multipotency. A similar phenotype was found in claudin-low breast cancer cells with properties of EMT and stemness. This intersection between EMT and stemness in TS cells and claudin-low metastatic breast cancer demonstrates the usefulness of developmental EMT systems to understand EMT in cancer.

We propose a new and effective statistical framework for identifying genome-wide differential changes in epigenetic marks with ChIP-seq data or gene expression with mRNA-seq data, and we develop a new software tool EpiCenter that can efficiently perform data analysis. The key features of our framework are: (i) providing multiple normalization methods to achieve appropriate normalization under different scenarios, (ii) using a sequence of three statistical tests to eliminate background regions and to account for different sources of variation and (iii) allowing adjustment for multiple testing to control false discovery rate (FDR) or family-wise type I error. Our software EpiCenter can perform multiple analytic tasks including: (i) identifying genome-wide epigenetic changes or differentially expressed genes, (ii) finding transcription factor binding sites and (iii) converting multiple-sample sequencing data into a single read-count data matrix. By simulation, we show that our framework achieves a low FDR consistently over a broad range of read coverage and biological variation. Through two real examples, we demonstrate the effectiveness of our framework and the usages of our tool. In particular, we show that our novel and robust ‘parsimony’ normalization method is superior to the widely-used ‘tagRatio’ method. Our software EpiCenter is freely available to the public.

Trophoblast differentiation during placentation involves an epithelial-mesenchymal transition (EMT) with loss of E-cadherin and gain of trophoblast invasiveness. Mice harboring a point mutation that renders inactive the mitogen-activated protein kinase kinase kinase MEKK4 exhibit dysregulated placental development with increased trophoblast invasion. Isolated MEKK4 kinase-inactive trophoblast stem (TS) cells cultured under undifferentiating, self-renewing conditions in the presence of fibroblast growth factor 4 (FGF4) display increased expression of Slug, Twist, and matrix metalloproteinase 2 (MMP2), loss of E-cadherin, and hyperinvasion of extracellular matrix, each a hallmark of EMT. MEKK4 kinase-inactive TS cells show a preferential differentiation to Tpbp alpha- and Gcm1-positive trophoblasts, which are indicative of spongiotrophoblast and syncytiotrophoblast differentiation, respectively. FGF4-stimulated Jun N-terminal kinase (JNK) and p38 activity is markedly reduced in MEKK4 kinase-inactive TS cells. Chemical inhibition of JNK in wild-type TS cells induced a similar EMT response as loss of MEKK4 kinase activity, including inhibition of E-cadherin expression and increased expression of Slug, MMP2, Tpbp alpha, and Gcm1. Chromatin immunoprecipitation analyses revealed changes in AP-1 composition with increased Fra-2 and decreased Fra-1 and JunB binding to the regulatory regions of Gcm1 and MMP2 genes in MEKK4 kinase-inactive TS cells. Our results define MEKK4 as a signaling hub for FGF4 activation of JNK that is required for maintenance of TS cells in an undifferentiated state.

During brain development, proper neuronal migration and morphogenesis is critical for the establishment of functional neural circuits. Here we report that srGAP2 negatively regulates neuronal migration and induces neurite outgrowth and branching through the ability of its F-BAR domain to induce filopodia-like membrane protrusions resembling those induced by I-BAR domains in vivo and in vitro. Previous work has suggested that in non-neuronal cells, filopodia dynamics decreases the rate of cell migration and the persistence of leading edge protrusions. srGAP2 knockdown reduces leading process branching and increases the rate of neuronal migration in vivo. Overexpression of srGAP2 or its F-BAR domain has the opposite effects, increasing leading process branching and decreasing migration. These results (1) suggest that F-BAR domains are functionally diverse and (2) highlight the functional importance of proteins directly regulating membrane deformation for proper neuronal migration and morphogenesis.