SKOR1 mediates FER kinase-dependent invasive growth of breast cancer cells.

High expression of the non-receptor tyrosine kinase FER is an independent prognostic factor that correlates with poor survival in breast cancer patients. To investigate whether the kinase activity of FER is essential for its oncogenic properties, we developed an ATP analogue-sensitive knock-in allele (FERASKI). Specific FER kinase inhibition in MDA-MB-231 cells reduces migration and invasion, as well as metastasis when xenografted into a mouse model of breast cancer. Using the FERASKI system, we identified Ski family transcriptional corepressor 1 (SKOR1) as a direct FER kinase substrate. SKOR1 loss phenocopies FER inhibition, leading to impaired proliferation, migration and invasion, and inhibition of breast cancer growth and metastasis formation in mice. We show that SKOR1 Y234, a candidate FER phosphorylation site, is essential for FER-dependent tumor progression. Finally, our work suggests that the SKOR1 Y234 residue promotes Smad2/3 signaling through SKOR1 binding to Smad3. Our study thus identifies SKOR1 as a mediator of FER-dependent progression of high-risk breast cancers.

Gastrointestinal cancer-associated fibroblasts expressing Junctional Adhesion Molecule-A are amenable to infection by oncolytic reovirus.

Gastrointestinal (GI) cancers are characterized by extensive tumor stroma that both promotes tumor progression and acts as a physical barrier for adjacent tumor cells, limiting the effect of current treatment modalities. Oncolytic virotherapy is currently investigated in clinical trials as a novel therapeutic agent for different malignancies of the GI tract, but it is largely unknown whether these viruses can also target the tumor stroma. Here, we investigated the tropism of two commonly studied OVs, adenovirus and reovirus, towards primary GI fibroblasts from human oesophageal, gastric, duodenal and pancreatic carcinomas (N = 36). GI fibroblasts were susceptible to type 3 Dearing (T3D) strain R124 and bioselected mutant reovirus (jin-3) infection but not oncolytic adenovirus (Ad5-Δ24). Efficient infection and apoptosis of human and mouse GI cancer-derived fibroblasts by these reoviruses was partially dependent on the expression of the reovirus entry receptor, Junctional Adhesion Molecule-A (JAM-A). Moreover, human GI cancer organoid-fibroblast co-cultures showed higher overall infectivity when containing JAM-A expressing fibroblasts as compared to JAM-A negative fibroblasts, indicating a potential role of JAM-A expressing fibroblasts for viral dissemination. We further show that JAM-A is not only necessary for efficient reovirus infection of fibroblasts but also partially mediates reovirus-induced apoptosis, dependent on signaling through the C-terminal PDZ-domain of JAM-A. Altogether, our data show the presence of JAM-A expressing fibroblasts in both human and murine GI cancers that are amenable to infection and induction of apoptosis by reovirus, extending the potential anti-cancer actions of reovirus with stromal targeting.

Dynamic Visualization of TGF-ß/SMAD3 Transcriptional Responses in Single Living Cells.

Transforming growth factor-ß (TGF-ß) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-ß/SMAD3 signaling kinetics, we developed a dynamic TGF-ß/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-ß/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-ß challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-ß/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.

Nonhuman Primate Adenoviruses of the Human Adenovirus B Species Are Potent and Broadly Acting Oncolytic Vector Candidates.

The use of human adenoviruses (hAds) as oncolytic agents has demonstrated considerable potential. However, their efficacy in clinical studies is generally moderate and often varies between patients. This may, in part, be attributable to variable pre-existing neutralizing immunity in patients, which can impact the antitumor efficacy and lead to response heterogeneity. Our aim was to isolate new Ads for the development of oncolytic vectors with low prevalence of neutralizing immunity in the human population. To this end, we isolated a collection of new nonhuman primate (nhp) Ads from stool samples of four great ape species held captive. We elected 12 isolates comprising the broadest genetic variability for further characterization. For three new nhpAds, all classified as the human adenovirus B (HAdV-B) species, no neutralizing activity could be detected when exposed to a preparation of immunoglobulins isolated from a pool of >1,000 donors as a surrogate of population immunity. In addition, the nhpAds of the HAdV-B species showed enhanced oncolytic potency compared to nhpAds of the HAdV-C species as well as to human adenovirus type 5 (HAdV-C5) in vitro when tested in a panel of 29 human cancer cell lines. Next-generation sequencing of the viral genomes revealed higher sequence similarity between hAds and nhpAds of HAdV-B compared to HAdV-C, which might underlie the differences in oncolytic ability. As a proof-of-concept, the Rb-binding domain of the E1A protein of the gorilla-derived HAdV-B nhpAd-lumc007 was deleted, thereby creating a new oncolytic derivative, which demonstrated increased oncolytic potential compared to HAdV-C5. Collectively, our data demonstrate that nhpAds of the HAdV-B species can serve as an alternative for the development of potent oncolytic Ad vectors with limited pre-existing neutralizing immunity in humans.

Metabolic Reprogramming of Mammary Epithelial Cells during TGF-ß-Induced Epithelial-to-Mesenchymal Transition.

The cytokine transforming growth factor-ß (TGF-ß) can induce normal breast epithelial cells to take on a mesenchymal phenotype, termed epithelial-to-mesenchymal transition (EMT). While the transcriptional and proteomic changes during TGF-ß-induced EMT have been described, the metabolic rewiring that occurs in epithelial cells undergoing EMT is not well understood. Here, we quantitively analyzed the TGF-ß-induced metabolic reprogramming during EMT of non-transformed NMuMG mouse mammary gland epithelial cells using nuclear magnetic resonance (NMR) spectroscopy. We found that TGF-ß elevates glycolytic and tricarboxylic acid (TCA)-cycle activity and increases glutaminolysis. Additionally, TGF-ß affects the hexosamine pathway, arginine-proline metabolism, the cellular redox state, and strongly affects choline metabolism during EMT. TGF-ß was found to induce phosphocholine production. A kinase inhibitor RSM-93A that inhibits choline kinase α (CHKα) mitigated TGF-ß-induced changes associated with EMT, i.e., increased filamentous (F)-actin stress fiber formation and N-Cadherin mesenchymal marker expression.

A FOXO-dependent replication checkpoint restricts proliferation of damaged cells.

DNA replication is challenged by numerous exogenous and endogenous factors that can interfere with the progression of replication forks. Substantial accumulation of single-stranded DNA during DNA replication activates the DNA replication stress checkpoint response that slows progression from S/G2 to M phase to protect genomic integrity. Whether and how mild replication stress restricts proliferation remains controversial. Here, we identify a cell cycle exit mechanism that prevents S/G2 phase arrested cells from undergoing mitosis after exposure to mild replication stress through premature activation of the anaphase promoting complex/cyclosome (APC/CCDH1). We find that replication stress causes a gradual decrease of the levels of the APC/CCDH1 inhibitor EMI1/FBXO5 through Forkhead box O (FOXO)-mediated inhibition of its transcription factor E2F1. By doing so, FOXOs limit the time during which the replication stress checkpoint is reversible and thereby play an important role in maintaining genomic stability.

TGFß-induced metabolic reprogramming during epithelial-to-mesenchymal transition in cancer.

Metastasis is the most frequent cause of death in cancer patients. Epithelial-to-mesenchymal transition (EMT) is the process in which cells lose epithelial integrity and become motile, a critical step for cancer cell invasion, drug resistance and immune evasion. The transforming growth factor-ß (TGFß) signaling pathway is a major driver of EMT. Increasing evidence demonstrates that metabolic reprogramming is a hallmark of cancer and extensive metabolic changes are observed during EMT. The aim of this review is to summarize and interconnect recent findings that illustrate how changes in glycolysis, mitochondrial, lipid and choline metabolism coincide and functionally contribute to TGFß-induced EMT. We describe TGFß signaling is involved in stimulating both glycolysis and mitochondrial respiration. Interestingly, the subsequent metabolic consequences for the redox state and lipid metabolism in cancer cells are found to be in favor of EMT as well. Combined we illustrate that a better understanding of the mechanistic links between TGFß signaling, cancer metabolism and EMT holds promising strategies for cancer therapy, some of which are already actively being explored in the clinic.

Assessment of Functional Phosphatidylinositol 3-Kinase Pathway Activity in Cancer Tissue Using Forkhead Box-O Target Gene Expression in a Knowledge-Based Computational Model.

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly activated in cancer. Tumors are potentially sensitive to PI3K pathway inhibitors, but reliable diagnostic tests that assess functional PI3K activity are lacking. Because PI3K pathway activity negatively regulates forkhead box-O (FOXO) transcription factor activity, FOXO target gene expression is inversely correlated with PI3K activity. A knowledge-based Bayesian computational model was developed to infer PI3K activity in cancer tissue samples from FOXO target gene mRNA levels and validated in cancer cell lines treated with PI3K inhibitors. However, applied to patient tissue samples, FOXO was often active in cancer types with expected active PI3K. SOD2 was differentially expressed between FOXO-active healthy and cancer tissue samples, indicating that cancer-associated cellular oxidative stress alternatively activated FOXO. To enable correct interpretation of active FOXO in cancer tissue, threshold levels for normal SOD2 expression in healthy tissue were defined above which FOXO activity is oxidative stress induced and below which PI3K regulated. In slow-growing luminal A breast cancer and low Gleason score prostate cancer, FOXO was active in a PI3K-regulated manner, indicating inactive PI3K. In aggressive luminal B, HER2, and basal breast cancer, FOXO was increasingly inactive or actively induced by oxidative stress, indicating PI3K activity. We provide a decision tree that facilitates functional PI3K pathway activity assessment in tissue samples from patients with cancer for therapy response prediction and prognosis.

FOXO Transcription Factors Both Suppress and Support Breast Cancer Progression.

FOXO transcription factors are regulators of cellular homeostasis and putative tumor suppressors, yet the role of FOXO in cancer progression remains to be determined. The data on FOXO function, particularly for epithelial cancers, are fragmentary and come from studies that focused on isolated aspects of cancer. To clarify the role of FOXO in epithelial cancer progression, we characterized the effects of inducible FOXO activation and loss in a mouse model of metastatic invasive lobular carcinoma. Strikingly, either activation or loss of FOXO function suppressed tumor growth and metastasis. We show that the multitude of cellular processes critically affected by FOXO function include proliferation, survival, redox homeostasis, and PI3K signaling, all of which must be carefully balanced for tumor cells to thrive.Significance: FOXO proteins are not solely tumor suppressors, but also support tumor growth and metastasis by regulating a multitude of cellular processes essential for tumorigenesis. Cancer Res; 78(9); 2356-69. ©2018 AACR.

Interplay between metabolic identities in the intestinal crypt supports stem cell function.

The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5+ crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt. Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5+ CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5+ CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5+ CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5+ CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5+ CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.

The Hallmarks of Cancer from a Redox Perspective.

SIGNIFICANCE: For a healthy cell to turn into a cancer cell and grow out to become a tumor, it needs to undergo a series of complex changes and acquire certain traits, summarized as "The Hallmarks of Cancer." These hallmarks can all be regarded as the result of altered signal transduction cascades and an understanding of these cascades is essential for cancer treatment. RECENT ADVANCES: Redox signaling is a long overlooked form of signal transduction that proceeds through the reversible oxidation of cysteines in proteins and that uses hydrogen peroxide as a second messenger. CRITICAL ISSUES: In this article, we provide examples that show that redox signaling is involved in the regulation of proteins and signaling cascades that play roles in every hallmark of cancer. FUTURE DIRECTIONS: An understanding of how redox signaling and "classical" signal transduction are intertwined could hold promising strategies for cancer therapy in the future. Antioxid. Redox Signal. 25, 300-325.

FOXOs support the metabolic requirements of normal and tumor cells by promoting IDH1 expression.

FOXO transcription factors are considered bona fide tumor suppressors; however, recent studies showed FOXOs are also required for tumor survival. Here, we identify FOXOs as transcriptional activators of IDH1. FOXOs promote IDH1 expression and thereby maintain the cytosolic levels of α-ketoglutarate and NADPH. In cancer cells carrying mutant IDH1, FOXOs likewise stimulate mutant IDH1 expression and maintain the levels of the oncometabolite 2-hydroxyglutarate, which stimulates cancer cell proliferation and inhibits TET enzymes and histone demethylases. Combined, our data provide a new paradigm for the paradoxical role of FOXOs in both tumor suppression and promotion.

Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells.

Animal mitotic spindle assembly relies on centrosome-dependent and centrosome-independent mechanisms, but their relative contributions remain unknown. Here, we investigated the molecular basis of the centrosome-independent spindle assembly pathway by performing a whole-genome RNAi screen in Drosophila S2 cells lacking functional centrosomes. This screen identified 197 genes involved in acentrosomal spindle assembly, eight of which had no previously described mitotic phenotypes and produced defective and/or short spindles. All 197 genes also produced RNAi phenotypes when centrosomes were present, indicating that none were entirely selective for the acentrosomal pathway. However, a subset of genes produced a selective defect in pole focusing when centrosomes were absent, suggesting that centrosomes compensate for this shape defect. Another subset of genes was specifically associated with the formation of multipolar spindles only when centrosomes were present. We further show that the chromosomal passenger complex orchestrates multiple centrosome-independent processes required for mitotic spindle assembly/maintenance. On the other hand, despite the formation of a chromosome-enriched RanGTP gradient, S2 cells depleted of RCC1, the guanine-nucleotide exchange factor for Ran on chromosomes, established functional bipolar spindles. Finally, we show that cells without functional centrosomes have a delay in chromosome congression and anaphase onset, which can be explained by the lack of polar ejection forces. Overall, these findings establish the constitutive nature of a centrosome-independent spindle assembly program and how this program is adapted to the presence/absence of centrosomes in animal somatic cells.

Mammary-specific inactivation of E-cadherin and p53 impairs functional gland development and leads to pleomorphic invasive lobular carcinoma in mice.

Breast cancer is the most common malignancy in women of the Western world. Even though a large percentage of breast cancer patients show pathological complete remission after standard treatment regimes, approximately 30-40% are non-responsive and ultimately develop metastatic disease. To generate a good preclinical model of invasive breast cancer, we have taken a tissue-specific approach to somatically inactivate p53 and E-cadherin, the cardinal cell-cell adhesion receptor that is strongly associated with tumor invasiveness. In breast cancer, E-cadherin is found mutated or otherwise functionally silenced in invasive lobular carcinoma (ILC), which accounts for 10-15% of all breast cancers. We show that mammary-specific stochastic inactivation of conditional E-cadherin and p53 results in impaired mammary gland function during pregnancy through the induction of anoikis resistance of mammary epithelium, resulting in loss of epithelial organization and a dysfunctional mammary gland. Moreover, combined inactivation of E-cadherin and p53 induced lactation-independent development of invasive and metastatic mammary carcinomas, which showed strong resemblance to human pleomorphic ILC. Dissemination patterns of mouse ILC mimic the human malignancy, showing metastasis to the gastrointestinal tract, peritoneum, lung, lymph nodes and bone. Our results confirm that loss of E-cadherin contributes to both mammary tumor initiation and metastasis, and establish a preclinical mouse model of human ILC that can be used for the development of novel intervention strategies to treat invasive breast cancer.