A glycolytic metabolite bypasses "two-hit" tumor suppression by BRCA2.

Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.

Comprehensive characterization of genomic features and clinical outcomes following targeted therapy and secondary cytoreductive surgery in OCCC: a single center experience.

OBJECTIVE: Ovarian clear cell carcinoma (OCCC) is associated with chemoresistance. Limited data exists regarding the efficacy of targeted therapies such as immune checkpoint inhibitors (ICI) and bevacizumab, and the role of secondary cytoreductive surgery (SCS). METHODS: We retrospectively analyzed genomic features and treatment outcomes of 172 OCCC patients treated at our institution from January 2000 to May 2022. Next-generation sequencing (NGS) was performed where sufficient archival tissue was available. RESULTS: 64.0% of patients were diagnosed at an early stage, and 36.0% at an advanced stage. Patients with advanced/relapsed OCCC who received platinum-based chemotherapy plus bevacizumab followed by maintenance bevacizumab had a median first-line progression-free survival (PFS) of 12.2 months, compared with 9.3 months for chemotherapy alone (hazard ratio=0.69; 95% confidence interval [CI]=0.33, 1.45). In 27 patients who received an ICI, the overall response rate was 18.5% and median duration of response was 7.4 months (95% CI=6.5, 8.3). In 17 carefully selected patients with fewer than 3 sites of relapse, median PFS was 35 months (95% CI=0, 73.5) and median overall survival was 96.8 months (95% CI=44.6, 149.0) after SCS. NGS on 58 tumors revealed common mutations in ARID1A (48.3%), PIK3CA (46.6%), and KRAS (20.7%). Pathogenic alterations in PIK3CA, FGFR2, and NBN were associated with worse survival outcomes. Median tumor mutational burden was 3.78 (range, 0-16). All 26 patients with available loss of heterozygosity (LOH) scores had LOH <16%. CONCLUSION: Our study demonstrates encouraging outcomes with bevacizumab and ICI, and SCS in select relapsed OCCC patients. Prospective trials are warranted.

SHP2 inhibitors maintain TGFß signalling through SMURF2 inhibition.

Despite the promising antitumor activity of SHP2 inhibitors in RAS-dependent tumours, overall responses have been limited by their narrow therapeutic window. Like with all MAPK pathway inhibitors, this is likely the result of compensatory pathway activation mechanisms. However, the underlying mechanisms of resistance to SHP2 inhibition remain unknown. The E3 ligase SMURF2 limits TGFß activity by ubiquitinating and targeting the TGFß receptor for proteosome degradation. Using a functional RNAi screen targeting all known phosphatases, we identify that the tyrosine phosphatase SHP2 is a critical regulator of TGFß activity. Specifically, SHP2 dephosphorylates two key residues on SMURF2, resulting in activation of the enzyme. Conversely, SHP2 depletion maintains SMURF2 in an inactive state, resulting in the maintenance of TGFß activity. Furthermore, we demonstrate that depleting SHP2 has significant implications on TGFß-mediated migration, senescence, and cell survival. These effects can be overcome through the use of TGFß-targeted therapies. Consequently, our findings provide a rationale for combining SHP2 and TGFß inhibitors to enhance tumour responses leading to improved patient outcomes.

Intrinsic cancer cell phosphoinositide 3-kinase δ regulates fibrosis and vascular development in cholangiocarcinoma.

BACKGROUND & AIMS: The class I- phosphatidylinositol-3 kinases (PI3Ks) signalling is dysregulated in almost all human cancers whereas the isoform-specific roles remain poorly investigated. We reported that the isoform δ (PI3Kδ) regulated epithelial cell polarity and plasticity and recent developments have heightened its role in hepatocellular carcinoma (HCC) and solid tumour progression. However, its role in cholangiocarcinoma (CCA) still lacks investigation. APPROACH & RESULTS: Immunohistochemical analyses of CCA samples reveal a high expression of PI3Kδ in the less differentiated CCA. The RT-qPCR and immunoblot analyses performed on CCA cells stably overexpressing PI3Kδ using lentiviral construction reveal an increase of mesenchymal and stem cell markers and the pluripotency transcription factors. CCA cells stably overexpressing PI3Kδ cultured in 3D culture display a thick layer of ECM at the basement membrane and a wide single lumen compared to control cells. Similar data are observed in vivo, in xenografted tumours established with PI3Kδ-overexpressing CCA cells in immunodeficient mice. The expression of mesenchymal and stemness genes also increases and tumour tissue displays necrosis and fibrosis, along with a prominent angiogenesis and lymphangiogenesis, as in mice liver of AAV8-based-PI3Kδ overexpression. These PI3Kδ-mediated cell morphogenesis and stroma remodelling were dependent on TGFß/Src/Notch signalling. Whole transcriptome analysis of PI3Kδ using the cancer cell line encyclopedia allows the classification of CCA cells according to cancer progression. CONCLUSIONS: Overall, our results support the critical role of PI3Kδ in the progression and aggressiveness of CCA via TGFß/src/Notch-dependent mechanisms and open new directions for the classification and treatment of CCA patients.

Histone Methyltransferase NSD2 Activates PKCα to Drive Metabolic Reprogramming and Lenalidomide Resistance in Multiple Myeloma.

Multiple myeloma cells undergo metabolic reprogramming in response to the hypoxic and nutrient-deprived bone marrow microenvironment. Primary oncogenes in recurrent translocations might be able to drive metabolic heterogeneity to survive the microenvironment that can present new vulnerabilities for therapeutic targeting. t(4;14) translocation leads to the universal overexpression of histone methyltransferase NSD2 that promotes plasma cell transformation through a global increase in H3K36me2. Here, we identified PKCα as an epigenetic target that contributes to the oncogenic potential of NSD2. RNA sequencing of t(4;14) multiple myeloma cell lines revealed a significant enrichment in the regulation of metabolic processes by PKCα, and the glycolytic gene, hexokinase 2 (HK2), was transcriptionally regulated by PKCα in a PI3K/Akt-dependent manner. Loss of PKCα displaced mitochondria-bound HK2 and reversed sensitivity to the glycolytic inhibitor 3-bromopyruvate. In addition, the perturbation of glycolytic flux led to a metabolic shift to a less energetic state and decreased ATP production. Metabolomics analysis indicated lactate as a differential metabolite associated with PKCα. As a result, PKCα conferred resistance to the immunomodulatory drugs (IMiD) lenalidomide in a cereblon-independent manner and could be phenocopied by either overexpression of HK2 or direct supplementation of lactate. Clinically, t(4;14) patients had elevated plasma lactate levels and did not benefit from lenalidomide-based regimens. Altogether, this study provides insights into the epigenetic-metabolism cross-talk in multiple myeloma and highlights the opportunity for therapeutic intervention that leverages the distinct metabolic program in t(4;14) myeloma. SIGNIFICANCE: Aberrant glycolysis driven by NSD2-mediated upregulation of PKCα can be therapeutically exploited using metabolic inhibitors with lactate as a biomarker to identify high-risk patients who exhibit poor response towards IMiD-based regimens.

SIRIUS, Ultra-Scintillating Upconversion Breast Implant for Remote Orthotopic Photodynamic Therapy.

Present day strategies for delivery of wireless photodynamic therapy (PDT) to deep-seated targets are limited by the inadequacy of irradiance and insufficient therapeutic depth. Here we report the design and preclinical validation of a flexible wireless upconversion nanoparticle (UCNP) implant (SIRIUS) that is capable of large field, high intensity illumination for PDT of deep-seated tumors. The implant achieves this by incorporating submicrometer core-shell-shell NaYF4 UCNPs into its design, which significantly enhances upconversion efficiency and mitigates light loss from surface quenching. We demonstrate the efficacy of SIRIUS UCNP implant mediated PDT in preclinical breast cancer disease models. In our in vitro experiments, SIRIUS directed 5-Aminolevulinic Acid (5-ALA) based wireless PDT leads to significant reactive oxygen species (ROS) generation and tumor apoptosis in hormonal receptor+/HER2+ (MCF7) and triple-negative (MDA-MB-231) breast cancer cell lines. In our in vivo rodent model, SIRIUS-driven PDT is shown to be significant in regressing tumors when applied to orthotopically inoculated breast tumors. Following successful preclinical validation, we also describe a clinical prototype of UCNP breast implant with potential dual cosmetic and onco-therapeutic functions. SIRIUS is an upconversion breast implant for wireless PDT that fulfils all the design prerequisites necessary for seamless clinical translation.

The effect of inhibition of receptor tyrosine kinase AXL on DNA damage response in ovarian cancer.

AXL is a receptor tyrosine kinase that is often overexpressed in cancers. It contributes to pathophysiology in cancer progression and therapeutic resistance, making it an emerging therapeutic target. The first-in-class AXL inhibitor bemcentinib (R428/BGB324) has been granted fast track designation by the U.S. Food and Drug Administration (FDA) in STK11-mutated advanced metastatic non-small cell lung cancer and was also reported to show selective sensitivity towards ovarian cancers (OC) with a Mesenchymal molecular subtype. In this study, we further explored AXL's role in mediating DNA damage responses by using OC as a disease model. AXL inhibition using R428 resulted in the increase of DNA damage with the concurrent upregulation of DNA damage response signalling molecules. Furthermore, AXL inhibition rendered cells more sensitive to the inhibition of ATR, a crucial mediator for replication stress. Combinatory use of AXL and ATR inhibitors showed additive effects in OC. Through SILAC co-immunoprecipitation mass spectrometry, we identified a novel binding partner of AXL, SAM68, whose loss in OC cells harboured phenotypes in DNA damage responses similar to AXL inhibition. In addition, AXL- and SAM68-deficiency or R428 treatment induced elevated levels of cholesterol and upregulated genes in the cholesterol biosynthesis pathway. There might be a protective role of cholesterol in shielding cancer cells against DNA damage induced by AXL inhibition or SMA68 deficiency.

Super-enhancer-driven TOX2 mediates oncogenesis in Natural Killer/T Cell Lymphoma.

BACKGROUND: Extranodal natural killer/T-cell lymphoma (NKTL) is an aggressive type of non-Hodgkin lymphoma with dismal outcome. A better understanding of disease biology and key oncogenic process is necessary for the development of targeted therapy. Super-enhancers (SEs) have been shown to drive pivotal oncogenes in various malignancies. However, the landscape of SEs and SE-associated oncogenes remain elusive in NKTL. METHODS: We used Nano-ChIP-seq of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs NKTL primary tumor samples. Integrative analysis of RNA-seq and survival data further pinned down high value, novel SE oncogenes. We utilized shRNA knockdown, CRISPR-dCas9, luciferase reporter assay, ChIP-PCR to investigate the regulation of transcription factor (TF) on SE oncogenes. Multi-color immunofluorescence (mIF) staining was performed on an independent cohort of clinical samples. Various function experiments were performed to evaluate the effects of TOX2 on the malignancy of NKTL in vitro and in vivo. RESULTS: SE landscape was substantially different in NKTL samples in comparison with normal tonsils. Several SEs at key transcriptional factor (TF) genes, including TOX2, TBX21(T-bet), EOMES, RUNX2, and ID2, were identified. We confirmed that TOX2 was aberrantly overexpressed in NKTL relative to normal NK cells and high expression of TOX2 was associated with worse survival. Modulation of TOX2 expression by shRNA, CRISPR-dCas9 interference of SE function impacted on cell proliferation, survival and colony formation ability of NKTL cells. Mechanistically, we found that RUNX3 regulates TOX2 transcription by binding to the active elements of its SE. Silencing TOX2 also impaired tumor formation of NKTL cells in vivo. Metastasis-associated phosphatase PRL-3 has been identified and validated as a key downstream effector of TOX2-mediated oncogenesis. CONCLUSIONS: Our integrative SE profiling strategy revealed the landscape of SEs, novel targets and insights into molecular pathogenesis of NKTL. The RUNX3-TOX2-SE-TOX2-PRL-3 regulatory pathway may represent a hallmark of NKTL biology. Targeting TOX2 could be a valuable therapeutic intervene for NKTL patients and warrants further study in clinic.

A single-cell atlas identifies pretreatment features of primary imatinib resistance in chronic myeloid leukemia.

Primary resistance to tyrosine kinase inhibitors (TKIs) is a significant barrier to optimal outcomes in chronic myeloid leukemia (CML), but factors contributing to response heterogeneity remain unclear. Using single-cell RNA (scRNA) sequencing, we identified 8 statistically significant features in pretreatment bone marrow, which correlated with either sensitivity (major molecular response or MMR) or extreme resistance to imatinib (eventual blast crisis [BC] transformation). Employing machine-learning, we identified leukemic stem cell (LSC) and natural killer (NK) cell gene expression profiles predicting imatinib response with >80% accuracy, including no false positives for predicting BC. A canonical erythroid-specifying (TAL1/KLF1/GATA1) regulon was a hallmark of LSCs from patients with MMR and was associated with erythroid progenitor [ERP] expansion in vivo (P < .05), and a 2- to 10-fold (6.3-fold in group A vs 1.09-fold in group C) erythroid over myeloid bias in vitro. Notably, ERPs demonstrated exquisite TKI sensitivity compared with myeloid progenitors (P < .001). These LSC features were lost with progressive resistance, and MYC- and IRF1-driven inflammatory regulons were evident in patients who progressed to transformation. Patients with MMR also exhibited a 56-fold expansion (P < .01) of a normally rare subset of hyperfunctional adaptive-like NK cells, which diminished with progressive resistance, whereas patients destined for BC accumulated inhibitory NKG2A+ NK cells favoring NK cell tolerance. Finally, we developed antibody panels to validate our scRNA-seq findings. These panels may be useful for prospective studies of primary resistance, and in assessing the contribution of predetermined vs acquired factors in TKI response heterogeneity.

Immune-Hot tumor features associated with recurrence in early-stage ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a distinct histotype of ovarian cancer, which usually presages a worse prognosis upon recurrence. Identifying patients at risk for relapse is an unmet need to improve outcomes. A retrospective cohort analysis of 195 early-stage OCCC patients diagnosed between January 2011 and December 2019 at National Taiwan University Hospital was conducted to identify prognostic factors for recurrence, progression-free survival (PFS) and overall survival (OS). Molecular profiling of tumors was performed in a case-controlled cohort matched for adjuvant therapy for biomarker discovery. Multivariate Cox proportional hazard model revealed that paclitaxel-based chemotherapy was associated with better PFS than nonpaclitaxel chemotherapy (HR = 0.19, P = .006). The addition of bevacizumab was associated with better PFS, compared to no bevacizumab (HR = 0.09, P = .02). Neither showed significant improvement in OS. Recurrence is associated with an Immune-Hot tumor feature (P = .03), the CTLA-4-high subtype (P = .01) and increased infiltration of immune cells in general. The Immune-Hot feature (HR = 3.39, P = .005) and the CTLA-4-high subtype (HR = 2.13, P = .059) were associated with worse PFS. Immune-Hot tumor features could prognosticate recurrence in early-stage OCCC.

Nuclear pCHK1 as a potential biomarker of increased sensitivity to ATR inhibition.

Excessive genomic instability coupled with abnormalities in DNA repair pathways induces high levels of 'replication stress' when cancer cells propagate. Rather than hampering cancer cell proliferation, novel treatment strategies are turning their attention towards targeting cell cycle checkpoint kinases (such as ATR, CHK1, WEE1, and others) along the DNA damage response and replicative stress response pathways, thereby allowing unrepaired DNA damage to be carried forward towards mitotic catastrophe and apoptosis. The selective ATR kinase inhibitor elimusertib (BAY 1895344) has demonstrated preclinical and clinical monotherapy activity; however, reliable predictive biomarkers of treatment benefit are still lacking. In this study, using gene expression profiling of 24 cell lines from different cancer types and in a panel of ovarian cancer cell lines, we found that nuclear-specific enrichment of checkpoint kinase 1 (CHK1) correlated with increased sensitivity to elimusertib. Using an advanced multispectral imaging system in subsequent cell line-derived xenograft specimens, we showed a trend between nuclear phosphorylated CHK1 (pCHK1) staining and increased sensitivity to the ATR inhibitor elimusertib, indicating the potential value of pCHK1 expression as a predictive biomarker of ATR inhibitor sensitivity. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Refining the Role of Pyruvate Dehydrogenase Kinases in Glioblastoma Development.

Glioblastoma (GB) are the most frequent brain cancers. Aggressive growth and limited treatment options induce a median survival of 12-15 months. In addition to highly proliferative and invasive properties, GB cells show cancer-associated metabolic characteristics such as increased aerobic glycolysis. Pyruvate dehydrogenase (PDH) is a key enzyme complex at the crossroads between lactic fermentation and oxidative pathways, finely regulated by PDH kinases (PDHKs). PDHKs are often overexpressed in cancer cells to facilitate high glycolytic flux. We hypothesized that targeting PDHKs, by disturbing cancer metabolic homeostasis, would alter GB progression and render cells vulnerable to additional cancer treatment. Using patient databases, distinct expression patterns of PDHK1 and PDHK2 in GB tissues were obvious. To disturb protumoral glycolysis, we modulated PDH activity through the genetic or pharmacological inhibition of PDHK in patient-derived stem-like spheroids. Striking effects of PDHKs inhibition using dichloroacetate were observed in vitro on cell morphology and metabolism, resulting in increased intracellular ROS levels and decreased proliferation and invasion. In vivo findings confirmed a reduction in tumor size and better survival of mice implanted with PDHK1 and PDHK2 knockout cells. Adding a radiotherapeutic protocol further resulted in a reduction in tumor size and improved mouse survival in our model.

PI3Kδ activity controls plasticity and discriminates between EMT and stemness based on distinct TGFß signaling.

The stem cells involved in formation of the complex human body are epithelial cells that undergo apicobasal polarization and form a hollow lumen. Epithelial plasticity manifests as epithelial to mesenchymal transition (EMT), a process by which epithelial cells switch their polarity and epithelial features to adopt a mesenchymal phenotype. The connection between the EMT program and acquisition of stemness is now supported by a substantial number of reports, although what discriminates these two processes remains largely elusive. In this study, based on 3D organoid culture of hepatocellular carcinoma (HCC)-derived cell lines and AAV8-based protein overexpression in the mouse liver, we show that activity modulation of isoform δ of phosphoinositide 3-kinase (PI3Kδ) controls differentiation and discriminates between stemness and EMT by regulating the transforming growth factor ß (TGFß) signaling. This study provides an important tool to control epithelial cell fate and represents a step forward in understanding the development of aggressive carcinoma.

TRAF4 Inhibits Bladder Cancer Progression by Promoting BMP/SMAD Signaling.

Patients with bladder cancer often have a poor prognosis due to the highly invasive and metastatic characteristics of bladder cancer cells. Epithelial-to-mesenchymal transition (EMT) has been causally linked to bladder cancer invasion. The E3 ubiquitin ligase, tumor necrosis factor receptor-associated factor 4 (TRAF4) has been implicated as a tumor promoter in a wide range of cancers. In contrast, here we show that low TRAF4 expression is associated with poor overall survival in patients with bladder cancer. We show that the TRAF4 gene is epigenetically silenced and that ERK mediates TRAF4 phosphorylation, resulting in lower TRAF4 protein levels in bladder cancer cells. In addition, we demonstrate that TRAF4 is inversely correlated with an EMT gene signature/protein marker expression. Functionally, by manipulating TRAF4 expression, we show that TRAF4 regulates EMT genes and epithelial and invasive properties in bladder cancer cells. Transcriptomic analysis of dysregulated TRAF4 expression in bladder cancer cell lines revealed that high TRAF4 expression enhances the bone morphogenetic protein (BMP)/SMAD and inhibits the NF-κB signaling pathway. Mechanistically, we show that TRAF4 targets the E3 ubiquitin ligase SMURF1, a negative regulator of BMP/SMAD signaling, for proteasomal degradation in bladder cancer cells. This was corroborated in patient samples where TRAF4 positively correlates with phospho-SMAD1/5, and negatively correlates with phospho-NFκb-p65. Lastly, we show that genetic and pharmacologic inhibition of SMURF1 inhibits the migration of aggressive mesenchymal bladder cancer cells. IMPLICATIONS: Our findings identify E3 ubiquitin ligase TRAF4 as a potential therapeutic target or biomarker for bladder cancer progression.

Osteopontin (OPN/SPP1), a Mediator of Tumor Progression, Is Regulated by the Mesenchymal Transcription Factor Slug/SNAI2 in Colorectal Cancer (CRC).

In colorectal cancer (CRC), disease-related death is closely linked to tumor aggressiveness and metastasis. Gene expression profiling of patient tumors has suggested that a more mesenchymal phenotype, present in about one-fourth of all patients, is associated with increased aggressiveness. Accordingly, the mesenchymal transcription factor Slug/SNAI2 has been associated with decreased disease-free survival. To decipher the basis for the Slug-mediated phenotype, we conducted RNAseq experiments with a panel of HT-29 CRC cells expressing different levels of Slug, both in vitro and in tumor models. The results show that osteopontin, a secreted pleotropic protein involved in multiple steps of colorectal cancer progression, was highly upregulated by Slug in vitro, as well as in vivo. We further show that Slug is a direct regulator of osteopontin at the promoter level. The levels of secreted osteopontin were correlated with Slug expression, thereby linking the tumor phenotype to a biomarker available by liquid biopsies. The results also suggest that osteopontin neutralization may attenuate at least some of the Slug-mediated functions.

BCG therapy downregulates HLA-I on malignant cells to subvert antitumor immune responses in bladder cancer.

Patients with high-risk, nonmuscle-invasive bladder cancer (NMIBC) frequently relapse after standard intravesical bacillus Calmette-Guérin (BCG) therapy and may have a dismal outcome. The mechanisms of resistance to such immunotherapy remain poorly understood. Here, using cancer cell lines, freshly resected human bladder tumors, and samples from cohorts of patients with bladder cancer before and after BCG therapy, we demonstrate 2 distinct patterns of immune subversion upon BCG relapse. In the first pattern, intracellular BCG infection of cancer cells induced a posttranscriptional downregulation of HLA-I membrane expression via inhibition of autophagy flux. Patients with HLA-I-deficient cancer cells following BCG therapy had a myeloid immunosuppressive tumor microenvironment (TME) with epithelial-mesenchymal transition (EMT) characteristics and dismal outcomes. Conversely, patients with HLA-I-proficient cancer cells after BCG therapy presented with CD8+ T cell tumor infiltrates, upregulation of inflammatory cytokines, and immune checkpoint-inhibitory molecules. The latter patients had a very favorable outcome. We surmise that HLA-I expression in bladder cancers at relapse following BCG does not result from immunoediting but rather from an immune subversion process directly induced by BCG on cancer cells, which predicts a dismal prognosis. HLA-I scoring of cancer cells by IHC staining can be easily implemented by pathologists in routine practice to stratify future treatment strategies for patients with urothelial cancer.

3D genome organization in the epithelial-mesenchymal transition spectrum.

BACKGROUND: The plasticity along the epithelial-mesenchymal transition (EMT) spectrum has been shown to be regulated by various epigenetic repertoires. Emerging evidence of local chromatin conformation changes suggests that regulation of EMT may occur at a higher order of three-dimensional genome level. RESULTS: We perform Hi-C analysis and combine ChIP-seq data across cancer cell lines representing different EMT states. We demonstrate that the epithelial and mesenchymal genes are regulated distinctively. We find that EMT genes are regulated within their topologically associated domains (TADs), with only a subset of mesenchymal genes being influenced by A/B compartment switches, indicating topological remodeling is required in the transcriptional regulation of these genes. At the TAD level, epithelial and mesenchymal genes are associated with different regulatory trajectories. The epithelial gene-residing TADs are enriched with H3K27me3 marks in the mesenchymal-like states. The mesenchymal gene-residing TADs, which do not show enrichment of H3K27me3 in epithelial-like states, exhibit increased interaction frequencies with regulatory elements in the mesenchymal-like states. CONCLUSIONS: We propose a novel workflow coupling immunofluorescence and dielectrophoresis to unravel EMT heterogeneity at single-cell resolution. The predicted three-dimensional structures of chromosome 10, harboring Vimentin, identify cell clusters of different states. Our results pioneer a novel avenue to decipher the complexities underlying the regulation of EMT and may infer the barriers of plasticity in the 3D genome context.

The H. pylori CagA Oncoprotein Induces DNA Double Strand Breaks through Fanconi Anemia Pathway Downregulation and Replication Fork Collapse.

The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

MicroRNA-196a promotes renal cancer cell migration and invasion by targeting BRAM1 to regulate SMAD and MAPK signaling pathways.

Rationale: MicroRNAs (miRNAs) are endogenous ~22nt RNAs that play critical regulatory roles in various biological and pathological processes, including various cancers. Their function in renal cancer has not been fully elucidated. It has been reported that miR-196a can act as oncogenes or as tumor suppressors depending on their target genes. However, the molecular target for miR-196a and the underlying mechanism in miR-196a promoted cell migration and invasion in renal cancer is still not clear. Methods: The expression, survival and correlation between miR-196a and BRAM1 were investigated using TCGA analysis and validated by RT-PCR and western blot. To visualize the effect of Bram1 on tumor metastasis in vivo, NOD-SCID gamma (NSG) mice were intravenously injected with RCC4 cells (106 cells/mouse) or RCC4 overexpressing Bram1. In addition, cell proliferation assays, migration and invasion assays were performed to examine the role of miR-196a in renal cells in vitro. Furthermore, immunoprecipitation was done to explore the binding targets of Bram1. Results: TCGA gene expression data from renal clear cell carcinoma patients showed a lower level of Bram1 expression in patients' specimens compared to adjacent normal tissues. Moreover, Kaplan­Meier survival data clearly show that high expression of Bram1correlates to poor prognosis in renal carcinoma patients. Our mouse metastasis model confirmed that Bram1 overexpression resulted in an inhibition in tumor metastasis. Target-prediction analysis and dual-luciferase reporter assay demonstrated that Bram1 is a direct target of miR-196a in renal cells. Further, our in vitro functional assays revealed that miR-196a promotes renal cell proliferation, migration, and invasion. Rescue of Bram1 expression reversed miR-196a-induced cell migration. MiR-196a promotes renal cancer cell migration by directly targeting Bram1 and inhibits Smad1/5/8 phosphorylation and MAPK pathways through BMPR1A and EGFR. Conclusions: Our findings thus provide a new mechanism on the oncogenic role of miR-196a and the tumor-suppressive role of Bram1 in renal cancer cells. Dysregulated miR-196a and Bram1 represent potential prognostic biomarkers and may have therapeutic applications in renal cancer.