IFI16-dependent STING signaling is a crucial regulator of anti-HER2 immune response in HER2+ breast cancer.

Relapse to anti-HER2 monoclonal antibody (mAb) therapies, such as trastuzumab in HER2+ breast cancer (BC), is associated with residual disease progression due to resistance to therapy. Here, we identify interferon-γ inducible protein 16 (IFI16)-dependent STING signaling as a significant determinant of trastuzumab responses in HER2+ BC. We show that down-regulation of immune-regulated genes (IRG) is specifically associated with poor survival of HER2+, but not other BC subtypes. Among IRG, IFI16 is identified as a direct target of EZH2, the underexpression of which leads to deficient STING activation and downstream CXCL10/11 expression in response to trastuzumab treatment. Dual inhibition of EZH2 and histone deacetylase (HDAC) significantly activates IFI16-dependent immune responses to trastuzumab. Notably, a combination of a novel histone methylation inhibitor with an HDAC inhibitor induces complete tumor eradication and long-term T cell memory in a HER2+ BC mouse model. Our findings demonstrate an epigenetic regulatory mechanism suppressing the expression of the IFI16-CXCL10/11 signaling pathway that provides a survival advantage to HER2+ BC to confer resistance to trastuzumab treatment.

Deep transcriptome profiling reveals limited conservation of A-to-I RNA editing in Xenopus.

BACKGROUND: Xenopus has served as a valuable model system for biomedical research over the past decades. Notably, ADAR was first detected in frog oocytes and embryos as an activity that unwinds RNA duplexes. However, the scope of A-to-I RNA editing by the ADAR enzymes in Xenopus remains underexplored. RESULTS: Here, we identify millions of editing events in Xenopus with high accuracy and systematically map the editome across developmental stages, adult organs, and species. We report diverse spatiotemporal patterns of editing with deamination activity highest in early embryogenesis before zygotic genome activation and in the ovary. Strikingly, editing events are poorly conserved across different Xenopus species. Even sites that are detected in both X. laevis and X. tropicalis show largely divergent editing levels or developmental profiles. In protein-coding regions, only a small subset of sites that are found mostly in the brain are well conserved between frogs and mammals. CONCLUSIONS: Collectively, our work provides fresh insights into ADAR activity in vertebrates and suggest that species-specific editing may play a role in each animal's unique physiology or environmental adaptation.

Multi-region sampling with paired sample sequencing analyses reveals sub-groups of patients with novel patient-specific dysregulation in Hepatocellular Carcinoma.

BACKGROUND: Conventional differential expression (DE) testing compares the grouped mean value of tumour samples to the grouped mean value of the normal samples, and may miss out dysregulated genes in small subgroup of patients. This is especially so for highly heterogeneous cancer like Hepatocellular Carcinoma (HCC). METHODS: Using multi-region sampled RNA-seq data of 90 patients, we performed patient-specific differential expression testing, together with the patients' matched adjacent normal samples. RESULTS: Comparing the results from conventional DE analysis and patient-specific DE analyses, we show that the conventional DE analysis omits some genes due to high inter-individual variability present in both tumour and normal tissues. Dysregulated genes shared in small subgroup of patients were useful in stratifying patients, and presented differential prognosis. We also showed that the target genes of some of the current targeted agents used in HCC exhibited highly individualistic dysregulation pattern, which may explain the poor response rate. DISCUSSION/CONCLUSION: Our results highlight the importance of identifying patient-specific DE genes, with its potential to provide clinically valuable insights into patient subgroups for applications in precision medicine.

Hypoxic tumor microenvironment activates GLI2 via HIF-1α and TGF-ß2 to promote chemoresistance in colorectal cancer.

Colorectal cancer patients often relapse after chemotherapy, owing to the survival of stem or progenitor cells referred to as cancer stem cells (CSCs). Although tumor stromal factors are known to contribute to chemoresistance, it remains not fully understood how CSCs in the hypoxic tumor microenvironment escape the chemotherapy. Here, we report that hypoxia-inducible factor (HIF-1α) and cancer-associated fibroblasts (CAFs)-secreted TGF-ß2 converge to activate the expression of hedgehog transcription factor GLI2 in CSCs, resulting in increased stemness/dedifferentiation and intrinsic resistance to chemotherapy. Genetic or small-molecule inhibitor-based ablation of HIF-1α/TGF-ß2-mediated GLI2 signaling effectively reversed the chemoresistance caused by the tumor microenvironment. Importantly, high expression levels of HIF-1α/TGF-ß2/GLI2 correlated robustly with the patient relapse following chemotherapy, highlighting a potential biomarker and therapeutic target for chemoresistance in colorectal cancer. Our study thus uncovers a molecular mechanism by which hypoxic colorectal tumor microenvironment promotes cancer cell stemness and resistance to chemotherapy and suggests a potentially targeted treatment approach to mitigating chemoresistance.

Albuminuria in Pediatric Patients With Adenotonsillar Hypertrophy.

The relationship between intermittent nocturnal hypoxia and albuminuria in pediatric patients with adenotonsillar hypertrophy was evaluated in this prospective study. Fifty children with grade 3 to 4 adenotonsillar hypertrophy scheduled for adenoidectomy and/or adenotonsillectomy were selected for study group. Fifteen patients with adenotonsillar grade 1 to 2 or adenotonsillectomized subjects in a similar range of sex, age, and body mass index scheduled for other surgeries were selected for control group. All children were monitored using finger pulse oximeter during the night before surgery. At the day of surgery, first morning urine samples were sent to the laboratory for analysis of albuminuria.Albuminuria was determined in 8 (16%) patients in study group and 1 (6.7%) patient in control group. This difference between groups was determined not to be statistically significant (P > 0.05). None of the pulse oximetry parameters was also found to be associated with albuminuria statistically (P > 0.05). However, the adenoid grade was observed to be associated with albuminuria (P = 0.011).This study revealed no relationship between albuminuria and intermittent hypoxia in children, although previous studies have reported that intermittent hypoxia causes albuminuria in adults. On the contrary, the adenoid grade was found to be in association with albuminuria. The reason seems to be unclear because of the lack of studies investigating albuminuria in children with adenotonsillar hypertrophy. However, inflammatory mediators arising from adenoid tissue may cause increase in renal capillary permeability and urine albumin excretion.

Is automated kinetic measurement superior to end-point for advanced oxidation protein product?

BACKGROUND: Advanced oxidation protein product (AOPP) was first described as an oxidative protein marker in chronic uremic patients and measured with a semi-automatic end-point method. Subsequently, the kinetic method was introduced for AOPP assay. We aimed to compare these two methods by adapting them to a chemistry analyzer and to investigate the correlation between AOPP and fibrinogen, the key molecule responsible for human plasma AOPP reactivity, microalbumin, and HbA1c in patients with type II diabetes mellitus (DM II). The effects of EDTA and citrate-anticogulated tubes on these two methods were incorporated into the study. METHODS: This study included 93 DM II patients (36 women, 57 men) with HbA1c levels > or = 7%, who were admitted to the diabetes and nephrology clinics. The samples were collected in EDTA and in citrate-anticoagulated tubes. Both methods were adapted to a chemistry analyzer and the samples were studied in parallel. RESULTS: In both types of samples, we found a moderate correlation between the kinetic and the endpoint methods (r = 0.611 for citrate-anticoagulated, r = 0.636 for EDTA-anticoagulated, p = 0.0001 for both). We found a moderate correlation between fibrinogen-AOPP and microalbumin-AOPP levels only in the kinetic method (r = 0.644 and 0.520 for citrate-anticoagulated; r = 0.581 and 0.490 for EDTA-anticoagulated, p = 0.0001). CONCLUSIONS: We conclude that adaptation of the end-point method to automation is more difficult and it has higher between-run CV% while application of the kinetic method is easier and it may be used in oxidative stress studies.

Simulated lunar microgravity transiently arrests growth and induces osteocyte-chondrocyte lineage differentiation in human Wharton's jelly stem cells.

Human Wharton's jelly stem cells (hWJSCs) are multipotent stem cells that are extensively employed in biotechnology applications. However, the impact of simulated lunar microgravity (sµG) on the growth, differentiation, and viability of this cell population is incompletely characterized. We aimed to determine whether acute (72 h) exposure to sµG elicited changes in growth and lineage differentiation in hWJSCs and if putative changes were maintained once exposure to terrestrial gravity (1.0 G) was restored. hWJSCs were cultured under standard 1.0 G conditions prior to being passaged and cultured under sµG (0.16 G) using a random positioning machine. Relative to control, hWJSCs cultured under sµG exhibited marked reductions in growth but not viability. Cell population expression of characteristic stemness markers (CD 73, 90, 105) was significantly reduced under sµG conditions. hWJSCs had 308 significantly upregulated and 328 significantly downregulated genes when compared to 1.0 G culture conditions. Key markers of cell replication, including MKI67, were inhibited. Significant upregulation of osteocyte-chondrocyte lineage markers, including SERPINI1, MSX2, TFPI2, BMP6, COMP, TMEM119, LUM, HGF, CHI3L1 and SPP1, and downregulation of cell fate regulators, including DNMT1 and EZH2, were detected in sµG-exposed hWJSCs. When returned to 1.0 G for 3 days, sµG-exposed hWJSCs had accelerated growth, and expression of stemness markers increased, approaching normal (i.e. 95%) levels. Our data support earlier findings that acute sµG significantly reduces the cell division potential of hWJSCs and suggest that acute sµG-exposure induces reversible changes in cell growth accompanied by osteocyte-chondrocyte changes in lineage differentiation.

Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy.

The hypoxic tumor microenvironment has been implicated in immune escape, but the underlying mechanism remains elusive. Using an in vitro culture system modeling human T cell dysfunction and exhaustion in triple-negative breast cancer (TNBC), we find that hypoxia suppresses immune effector gene expression, including in T and NK cells, resulting in immune effector cell dysfunction and resistance to immunotherapy. We demonstrate that hypoxia-induced factor 1α (HIF1α) interaction with HDAC1 and concurrent PRC2 dependency causes chromatin remolding resulting in epigenetic suppression of effector genes and subsequent immune dysfunction. Targeting HIF1α and the associated epigenetic machinery can reverse the immune effector dysfunction and overcome resistance to PD-1 blockade, as demonstrated both in vitro and in vivo using syngeneic and humanized mice models. These findings identify a HIF1α-mediated epigenetic mechanism in immune dysfunction and provide a potential strategy to overcome immune resistance in TNBC.

Molecular docking-aided identification of small molecule inhibitors targeting ß-catenin-TCF4 interaction.

Here we report a molecular docking-based approach to identify small molecules that can target the ß-catenin (ß-cat)-TCF4 protein-protein interaction (PPI), a key effector complex for nuclear Wnt signaling activity. Specifically, we developed and optimized a computational model of ß-cat using publicly available ß-cat protein crystal structures, and existing ß-cat-TCF4 interaction inhibitors as the training set. Using our computational model to an in silico screen predicted 27 compounds as good binders to ß-cat, of which 3 were identified to be effective against a Wnt-responsive luciferase reporter. In vitro functional validation experiments revealed GB1874 as an inhibitor of the Wnt pathway that targets the ß-cat-TCF4 PPI. GB1874 also affected the proliferation and stemness of Wnt-addicted colorectal cancer (CRC) cells in vitro. Encouragingly, GB1874 inhibited the growth of CRC tumor xenografts in vivo, thus demonstrating its potential for further development into therapeutics against Wnt-associated cancer indications.

Stromal induction of BRD4 phosphorylation Results in Chromatin Remodeling and BET inhibitor Resistance in Colorectal Cancer.

BRD4, a Bromodomain and Extraterminal (BET) protein family member, is a promising anti-cancer drug target. However, resistance to BET inhibitors targeting BRD4 is common in solid tumors. Here, we show that cancer-associated fibroblast (CAF)-activated stromal signaling, interleukin-6/8-JAK2, induces BRD4 phosphorylation at tyrosine 97/98 in colorectal cancer, resulting in BRD4 stabilization due to interaction with the deubiquitinase UCHL3. BRD4 phosphorylation at tyrosine 97/98 also displays increased binding to chromatin but reduced binding to BET inhibitors, resulting in resistance to BET inhibitors. We further show that BRD4 phosphorylation promotes interaction with STAT3 to induce chromatin remodeling through concurrent binding to enhancers and super-enhancers, supporting a tumor-promoting transcriptional program. Inhibition of IL6/IL8-JAK2 signaling abolishes BRD4 phosphorylation and sensitizes BET inhibitors in vitro and in vivo. Our study reveals a stromal mechanism for BRD4 activation and BET inhibitor resistance, which provides a rationale for developing strategies to treat CRC more effectively.

Patient-Specific Hemodynamics of New Coronary Artery Bypass Configurations.

PURPOSE: This study aims to quantify the patient-specific hemodynamics of complex conduit routing configurations of coronary artery bypass grafting (CABG) operation which are specifically suitable for off-pump surgeries. Coronary perfusion efficacy and local hemodynamics of multiple left internal mammary artery (LIMA) with sequential and end-to-side anastomosis are investigated. Using a full anatomical model comprised of aortic arch and coronary artery branches the optimum perfusion configuration in multi-vessel coronary artery stenosis is desired. METHODOLOGY: Two clinically relevant CABG configurations are created using a virtual surgical planning tool where for each configuration set, the stenosis level, anastomosis distance and angle were varied. A non-Newtonian computational fluid dynamics solver in OpenFOAM incorporated with resistance boundary conditions representing the coronary perfusion physiology was developed. The numerical accuracy is verified and results agreed well with a validated commercial cardiovascular flow solver and experiments. For segmental performance analysis, new coronary perfusion indices to quantify deviation from the healthy scenario were introduced. RESULTS: The first simulation configuration set;-a CABG targeting two stenos sites on the left anterior descending artery (LAD), the LIMA graft was capable of 31 mL/min blood supply for all the parametric cases and uphold the healthy LAD perfusion in agreement with the clinical experience. In the second end-to-side anastomosed graft configuration set;-the radial artery graft anastomosed to LIMA, a maximum of 64 mL/min flow rate in LIMA was observed. However, except LAD, the obtuse marginal (OM) and second marginal artery (m2) suffered poor perfusion. In the first set, average wall shear stress (WSS) were in the range of 4 to 35 dyns/cm2 for in LAD. Nevertheless, for second configuration sets the WSS values were higher as the LIMA could not supply enough blood to OM and m2. CONCLUSION: The virtual surgical configurations have the potential to improve the quality of operation by providing quantitative surgical insight. The degree of stenosis is a critical factor in terms of coronary perfusion and WSS. The sequential anastomosis can be done safely if the anastomosis angle is less than 90 degrees regardless of degree of stenosis. The smaller proposed perfusion index value, O(0.04 - 0) × 102, enable us to quantify the post-op hemodynamic performance by comparing with the ideal healthy physiological flow.

EZH2-mediated PP2A inactivation confers resistance to HER2-targeted breast cancer therapy.

HER2-targeted therapy has yielded a significant clinical benefit in patients with HER2+ breast cancer, yet disease relapse due to intrinsic or acquired resistance remains a significant challenge in the clinic. Here, we show that the protein phosphatase 2A (PP2A) regulatory subunit PPP2R2B is a crucial determinant of anti-HER2 response. PPP2R2B is downregulated in a substantial subset of HER2+ breast cancers, which correlates with poor clinical outcome and resistance to HER2-targeted therapies. EZH2-mediated histone modification accounts for the PPP2R2B downregulation, resulting in sustained phosphorylation of PP2A targets p70S6K and 4EBP1 which leads to resistance to inhibition by anti-HER2 treatments. Genetic depletion or inhibition of EZH2 by a clinically-available EZH2 inhibitor restores PPP2R2B expression, abolishes the residual phosphorylation of p70S6K and 4EBP1, and resensitizes HER2+ breast cancer cells to anti-HER2 treatments both in vitro and in vivo. Furthermore, the same epigenetic mechanism also contributes to the development of acquired resistance through clonal selection. These findings identify EZH2-dependent PPP2R2B suppression as an epigenetic control of anti-HER2 resistance, potentially providing an opportunity to mitigate anti-HER2 resistance with EZH2 inhibitors.

Effect of modified Blalock-Taussig shunt anastomosis angle and pulmonary artery diameter on pulmonary flow.

OBJECTIVE: This study aimed to identify the best graft-to-pulmonary artery (PA) anastomosis angle measuring pulmonary blood flow, wall shear stress (WSS), and shunt flow. METHODS: A tetralogy of Fallot with pulmonary atresia computer model was used to study three different modified Blalock-Taussig shunt (mBTS) anastomosis angle configurations with three different PA diameter configurations. Velocity and WSS were analyzed, and the flow rates at the right PA (RPA) and left PA (LPA) were calculated. RESULTS: A 4-mm and 8-mm diameter of RPA and LPA, respectively with vertical shunt angle produces the highest total flow. In the RPA larger diameter than the LPA configutations, the left-leaning shunt produces the lowest total PA flow whereas in the LPA larger diameter than the RPA configuratios, the right-leaning shunt produces the lowest total PA flow. Therefore, the shunt anastomosis should not be leaned through the narrow side of PA to reach best flow. As the flow inside the shunt increased, WSS also increased due to enhanced velocity gradients. CONCLUSION: The anastomosis angle between the conduit and PA affects the flow to PA. Vertical anastomosis configurations increase the total PA flow; thus, these configurations are preferable than the leaned configurations.

KDM4B-regulated unfolded protein response as a therapeutic vulnerability in PTEN-deficient breast cancer.

PTEN deficiency in breast cancer leads to resistance to PI3K-AKT inhibitor treatment despite aberrant activation of this signaling pathway. Here, we report that genetic depletion or small molecule inhibition of KDM4B histone demethylase activates the unfolded protein response (UPR) pathway and results in preferential apoptosis in PTEN-deficient triple-negative breast cancers (TNBCs). Intriguingly, this function of KDM4B on UPR requires its demethylase activity but is independent of its canonical role in histone modification, and acts through its cytoplasmic interaction with eIF2α, a crucial component of UPR signaling, resulting in reduced phosphorylation of this component. Targeting KDM4B in combination with PI3K inhibition induces further activation of UPR, leading to robust synergy in apoptosis. These findings identify KDM4B as a therapeutic vulnerability in PTEN-deficient TNBC that otherwise would be resistant to PI3K inhibition.

Chromosome 1q21.3 amplification is a trackable biomarker and actionable target for breast cancer recurrence.

Tumor recurrence remains the main reason for breast cancer-associated mortality, and there are unmet clinical demands for the discovery of new biomarkers and development of treatment solutions to benefit patients with breast cancer at high risk of recurrence. Here we report the identification of chromosomal copy-number amplification at 1q21.3 that is enriched in subpopulations of breast cancer cells bearing characteristics of tumor-initiating cells (TICs) and that strongly associates with breast cancer recurrence. Amplification is present in ∼10-30% of primary tumors but in more than 70% of recurrent tumors, regardless of breast cancer subtype. Detection of amplification in cell-free DNA (cfDNA) from blood is strongly associated with early relapse in patients with breast cancer and could also be used to track the emergence of tumor resistance to chemotherapy. We further show that 1q21.3-encoded S100 calcium-binding protein (S100A) family members, mainly S100A7, S100A8, and S100A9 (S100A7/8/9), and IL-1 receptor-associated kinase 1 (IRAK1) establish a reciprocal feedback loop driving tumorsphere growth. Notably, this functional circuitry can be disrupted by the small-molecule kinase inhibitor pacritinib, leading to preferential impairment of the growth of 1q21.3-amplified breast tumors. Our study uncovers the 1q21.3-directed S100A7/8/9-IRAK1 feedback loop as a crucial component of breast cancer recurrence, serving as both a trackable biomarker and an actionable therapeutic target for breast cancer.