Mutant mice with rod-specific VPS35 deletion exhibit retinal α-synuclein pathology-associated degeneration.

Vacuolar protein sorting 35 (VPS35), the core component of the retromer complex which regulates endosomal trafficking, is genetically linked with Parkinson's disease (PD). Impaired vision is a common non-motor manifestation of PD. Here, we show mouse retinas with VPS35-deficient rods exhibit synapse loss and visual deficit, followed by progressive degeneration concomitant with the emergence of Lewy body-like inclusions and phospho-α-synuclein (P-αSyn) aggregation. Ultrastructural analyses reveal VPS35-deficient rods accumulate aggregates in late endosomes, deposited as lipofuscins bound to P-αSyn. Mechanistically, we uncover a protein network of VPS35 and its interaction with HSC70. VPS35 deficiency promotes sequestration of HSC70 and P-αSyn aggregation in late endosomes. Microglia which engulf lipofuscins and P-αSyn aggregates are activated, displaying autofluorescence, observed as bright dots in fundus imaging of live animals, coinciding with pathology onset and progression. The Rod∆Vps35 mouse line is a valuable tool for further mechanistic investigation of αSyn lesions and retinal degenerative diseases.

Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression.

Low response rate, treatment relapse, and resistance remain key challenges for cancer treatment with immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TS) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Here we identify, by using in vivo CRISPR/Cas9 based loss-of-function screening, that NF1, TSC1, and TGF-ß RII as TSs regulating immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape, characterized by increased numbers of LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 and PD-L1 simultaneously inhibits metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-ß RII- deficient tumors. Our study thus reveals a role of TSs in regulating metastasis via non-cell-autonomous modulation of the immune compartment and provides proof-of-principle for ICB targeting LAG3 for patients with NF1-, TSC1- or TGF-ß RII-inactivated cancers.

Assessing the Correlation between Allostatic Load and False-Positive Image-Guided Breast Biopsies.

Background: Allostatic load (AL) is the accumulation of physiological dysregulation attributed to repeated activation of the stress response over a lifetime. We assessed the utility of AL as a prognostic measure for high-risk benign breast biopsy pathology results. Method: Eligible patients were women 18 years or older, with a false-positive outpatient breast biopsy between January and December 2022 at a tertiary academic health center. AL was calculated using 12 variables representing four physiological systems: cardiovascular (pulse rate, systolic and diastolic blood pressures, total cholesterol, high-density lipoprotein, and low-density lipoprotein); metabolic (body mass index, albumin, and hemoglobin A1C); renal (creatinine and estimated glomerular filtration rate); and immune (white blood cell count). Multivariable logistic regression was used to assess the association between AL before biopsy and breast biopsy outcomes controlling for patients' sociodemographics. Results: In total, 170 women were included (mean age, 54.1 ± 12.9 years): 89.4% had benign and 10.6% had high-risk pathologies (radial scar/complex sclerosing lesion, atypical ductal or lobular hyperplasia, flat epithelial atypia, intraductal papilloma, or lobular carcinoma in-situ). A total of 56.5% were White, 24.7% Asian, and 17.1% other races. A total of 32.5% identified as Hispanic. The mean breast cancer risk score using the Tyrer-Cuzick model was 11.9 ± 7.0. In multivariable analysis, with every one unit increase in AL, the probability of high-risk pathology increased by 37% (odds ratio, 1.37; 95% confidence interval, 1.03, 1.81; p = 0.03). No significant association was seen between high-risk pathology and age, ethnicity, breast cancer risk, or area deprivation index. Conclusion: Our findings support that increased AL, a biological marker of stress, is associated with high-risk pathology among patients with false-positive breast biopsy results.

Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer.

Patients with ER-negative breast cancer have the worst prognosis of all breast cancer subtypes, often experiencing rapid recurrence or progression to metastatic disease shortly after diagnosis. Given that metastasis is the primary cause of mortality in most solid tumors, understanding metastatic biology is crucial for effective intervention. Using a mouse systems genetics approach, we previously identified 12 genes associated with metastatic susceptibility. Here, we extend those studies to identify Resf1, a poorly characterized gene, as a novel metastasis susceptibility gene in ER- breast cancer. Resf1 is a large, unstructured protein with an evolutionarily conserved intron-exon structure, but with poor amino acid conservation. CRISPR or gene trap mouse models crossed to the Polyoma Middle-T antigen genetically engineered mouse model (MMTV-PyMT) demonstrated that reduction of Resf1 resulted in a significant increase in tumor growth, a shortened overall survival time, and increased incidence and number of lung metastases, consistent with patient data. Furthermore, an analysis of matched tail and primary tissues revealed loss of the wildtype copy in tumor tissue, consistent with Resf1 being a tumor suppressor. Mechanistic analysis revealed a potential role of Resf1 in transcriptional control through association with compound G4 quadruplexes in expressed sequences, particularly those associated with ribosomal biogenesis. These results suggest that loss of Resf1 enhances tumor progression in ER- breast cancer through multiple alterations in both transcriptional and translational control.

Detection and identification of single ribonucleotide monophosphates using a dual in-plane nanopore sensor made in a thermoplastic via replication.

We report the generation of ∼8 nm dual in-plane pores fabricated in a thermoplastic via nanoimprint lithography (NIL). These pores were connected in series with nanochannels, one of which served as a flight tube to allow the identification of single molecules based on their molecular-dependent apparent mobilities (i.e., dual in-plane nanopore sensor). Two different thermoplastics were investigated including poly(methyl methacrylate), PMMA, and cyclic olefin polymer, COP, as the substrate for the sensor both of which were sealed using a low glass transition cover plate (cyclic olefin co-polymer, COC) that could be thermally fusion bonded to the PMMA or COP substrate at a temperature minimizing nanostructure deformation. Unique to these dual in-plane nanopore sensors was two pores flanking each side of the nanometer flight tube (50 × 50 nm, width × depth) that was 10 µm in length. The utility of this dual in-plane nanopore sensor was evaluated to not only detect, but also identify single ribonucleotide monophosphates (rNMPs) by using the travel time (time-of-flight, ToF), the resistive pulse event amplitude, and the dwell time. In spite of the relatively large size of these in-plane pores (∼8 nm effective diameter), we could detect via resistive pulse sensing (RPS) single rNMP molecules at a mass load of 3.9 fg, which was ascribed to the unique structural features of the nanofluidic network and the use of a thermoplastic with low relative dielectric constants, which resulted in a low RMS noise level in the open pore current. Our data indicated that the identification accuracy of individual rNMPs was high, which was ascribed to an improved chromatographic contribution to the nano-electrophoresis apparent mobility. With the ToF data only, the identification accuracy was 98.3%. However, when incorporating the resistive pulse sensing event amplitude and dwell time in conjunction with the ToF and analyzed via principal component analysis (PCA), the identification accuracy reached 100%. These findings pave the way for the realization of a novel chip-based single-molecule RNA sequencing technology.

Loss of NAT10 disrupts enhancer organization via p300 mislocalization and suppresses transcription of genes necessary for metastasis progression.

Acetylation of protein and RNA represent a critical event for development and cancer progression. NAT10 is the only known RNA acetylase that catalyzes the N4-actylcytidine (ac4C) modification of RNAs. Here, we show that the loss of NAT10 significantly decreases lung metastasis in allograft and genetically engineered mouse models of breast cancer. NAT10 interacts with a mechanosensitive, metastasis susceptibility protein complex at the nuclear pore. In addition to its canonical role in RNA acetylation, we find that NAT10 interacts with p300 at gene enhancers. NAT10 loss is associated with p300 mislocalization into heterochromatin regions. NAT10 depletion disrupts enhancer organization, leading to alteration of gene transcription necessary for metastatic progression, including reduced myeloid cell-recruiting chemokines that results in a less metastasis-prone tumor microenvironment. Our study uncovers a distinct role of NAT10 in enhancer organization of metastatic tumor cells and suggests its involvement in the tumor-immune crosstalk dictating metastatic outcomes.

Establishment and Characterization of Amitrole-Induced Mouse Thyroid Adenomatous Nodule-Derived Cell Lines.

Background: Thyroid cancer cell lines have been of great value for the study of thyroid cancer. However, the availability of benign thyroid adenoma cell lines is limited. Methods: Cell lines were established from thyroid adenomatous nodules that developed in mice treated with the goitrogen amitrole. Expression of epithelial, mesenchymal, and thyroid markers of these established cell lines was determined, and the effect of lentivirus-transduced overexpression of NKX2-1, a master regulator of thyroid development, on the thyroid marker expression was examined. Signal transduction and cell proliferation were evaluated after treatment with insulin-like growth factor-I (IGF-I) and the selective IGF-I receptor (IGF-IR) inhibitor NVP-ADW742. Xenograft studies were performed to examine tumorigenicity of the cells in mice. Whole-genome sequencing (WGS) was used to comprehensively determine the genetic mutations in the established two cell lines. Results: Five mouse thyroid adenomatous nodules-derived cell lines named CAT (cells from amitrole-treated thyroids) were established. Among these, two cell lines, CAT458/458s (CAT458s: a subline of CAT458) and CAT459, were found to be positive for epithelial markers and negative for a mesenchymal marker. NKX2-1-positive CAT459 cells showed higher messenger RNA (mRNA) expression of some thyroid differentiation markers than NKX2-1-negative CAT458s cells, and NKX2-1 overexpression increased and/or induced their expression. IGF-I signaling was transduced in thyrotropin receptor (Tshr)-negative CAT458s and 459 cells, and NVP-ADW742 suppressed their proliferation. No tumors developed in mice after subcutaneous injection of CAT458s or 459 cells. The WGS analysis revealed the presence of missense mutations in the tumor suppressor genes such as Polk (encoding DNA polymerase kappa) and Tgfb1 (encoding transforming growth factor beta 1), while no mutations were found in the prominent thyroid cancer-related genes Braf, Trp53 (encoding p53), and Tert (encoding telomerase reverse transcriptase). Conclusions: Two mouse thyroid adenomatous nodule-derived cell lines with different thyroid differentiation marker expression were established. NKX2-1 induced partial differentiation of these cell lines. They lacked tumorigenicity and prominent gene mutations involved in thyroid cancer development, while missense mutations were found in some tumor suppressors as revealed by WGS. The CAT458s and 459 provide a new tool to further clarify the process of thyroid multistep carcinogenesis and differentiation.

SOX9 is a key component of RUNX2-regulated transcriptional circuitry in osteosarcoma.

BACKGROUND: The absence of prominent, actionable genetic alternations in osteosarcomas (OS) implies that transcriptional and epigenetic mechanisms significantly contribute to the progression of this life-threatening form of cancer. Therefore, the identification of potential transcriptional events that promote the survival of OS cells could be key in devising targeted therapeutic approaches for OS. We have previously shown that RUNX2 is a transcription factor (TF) essential for OS cell survival. Unfortunately, the transcriptional network or circuitry regulated by RUNX2 in OS cells is still largely unknown. METHODS: The TFs that are in the RUNX2 transcriptional circuitry were identified by analyzing RNAseq and ChIPseq datasets of RUNX2. To evaluate the effect of SOX9 knockdown on the survival of osteosarcoma cells in vitro, we employed cleaved caspase-3 immunoblotting and propidium iodide staining techniques. The impact of SOX9 and JMJD1C depletion on OS tumor growth was examined in vivo using xenografts and immunohistochemistry. Downstream targets of SOX9 were identified and dissected using RNAseq, pathway analysis, and gene set enrichment analysis. Furthermore, the interactome of SOX9 was identified using BioID and validated by PLA. RESULT: Our findings demonstrate that SOX9 is a critical TF that is induced by RUNX2. Both in vitro and in vivo experiments revealed that SOX9 plays a pivotal role in the survival of OS. RNAseq analysis revealed that SOX9 activates the transcription of MYC, a downstream target of RUNX2. Mechanistically, our results suggest a transcriptional network involving SOX9, RUNX2, and MYC, with SOX9 binding to RUNX2. Moreover, we discovered that JMJD1C, a chromatin factor, is a novel binding partner of SOX9, and depletion of JMJD1C impairs OS tumor growth. CONCLUSION: The findings of this study represent a significant advancement in our understanding of the transcriptional network present in OS cells, providing valuable insights that may contribute to the development of targeted therapies for OS.

Melanoma clonal subline analysis uncovers heterogeneity-driven immunotherapy resistance mechanisms.

Intratumoral heterogeneity (ITH) can promote cancer progression and treatment failure, but the complexity of the regulatory programs and contextual factors involved complicates its study. To understand the specific contribution of ITH to immune checkpoint blockade (ICB) response, we generated single cell-derived clonal sublines from an ICB-sensitive and genetically and phenotypically heterogeneous mouse melanoma model, M4. Genomic and single cell transcriptomic analyses uncovered the diversity of the sublines and evidenced their plasticity. Moreover, a wide range of tumor growth kinetics were observed in vivo , in part associated with mutational profiles and dependent on T cell-response. Further inquiry into melanoma differentiation states and tumor microenvironment (TME) subtypes of untreated tumors from the clonal sublines demonstrated correlations between highly inflamed and differentiated phenotypes with the response to anti-CTLA-4 treatment. Our results demonstrate that M4 sublines generate intratumoral heterogeneity at both levels of intrinsic differentiation status and extrinsic TME profiles, thereby impacting tumor evolution during therapeutic treatment. These clonal sublines proved to be a valuable resource to study the complex determinants of response to ICB, and specifically the role of melanoma plasticity in immune evasion mechanisms.

Newborn hearing screening methodology impacts the timing of diagnosis for auditory neuropathy spectrum disorder.

INTRODUCTION: Auditory Neuropathy Spectrum Disorder (ANSD) accounts for 10 % to 15 % of pediatric hearing loss. In most cases, otoacoustic emissions (OAE) are present as the outer hair cell function is normal, and the auditory brainstem response (ABR) is abnormal. Newborn hearing screen (NBHS) is completed using OAE or ABR depending on the institution. Because OAEs are often present in ANSD, NBHS done solely with OAE can miss and delay diagnosis of patients with ANSD. OBJECTIVES: To assess whether NBHS methodology impacts the age of diagnosis of ANSD. METHODS: This is a retrospective study of patients, 0-18 years of age, diagnosed with ANSD at two tertiary pediatric hospitals from 1/01/2010 to 12/31/2018 after referral from NBHS performed in the community. Data recorded included patient demographics, method of NBHS, NICU stay, and age at ANSD diagnosis. RESULTS: 264 patients were diagnosed with ANSD. Of those, 123 (46.6 %) were female, and 141 (53.4 %) were male. Ninety-seven (36.8 %) were admitted to NICU and the mean stay was 6.98 weeks (STD = 10.7; CI = 4.8-9.1). The majority (244, 92.4 %) of patients had NBHS with ABR, and 20 (7.5 %) had NBHS with OAE. Patients screened with ABR were diagnosed with ANSD earlier than those who screened with OAE, with a mean age of 14.1 versus 27.3 weeks (p = 0.0397, CI = 15.2-39.3). Among those screened with ABR, median age at diagnosis was 4 months for NICU infants and 2.5 months for infants with no history of NICU stay over 5 days. In comparison, median diagnosis age was 8 months for non-NICU infants screened with OAEs. CONCLUSION: Patients with ANSD who had NBHS with ABR were diagnosed earlier than those with OAE. Our data suggest that universal screening with ABR may facilitate earlier diagnosis of ANSD and earlier evaluation for aural rehabilitation, especially in high-risk cohorts such as NICU patients. Further research is needed into factors that contribute to earlier diagnosis among patients screened with ABR.

Apoptosis-induced nuclear expulsion in tumor cells drives S100a4-mediated metastatic outgrowth through the RAGE pathway.

Most tumor cells undergo apoptosis in circulation and at the metastatic organ sites due to host immune surveillance and a hostile microenvironment. It remains to be elucidated whether dying tumor cells have a direct effect on live tumor cells during the metastatic process and what the underlying mechanisms are. Here we report that apoptotic cancer cells enhance the metastatic outgrowth of surviving cells through Padi4-mediated nuclear expulsion. Tumor cell nuclear expulsion results in an extracellular DNA-protein complex that is enriched with receptor for advanced glycation endproducts (RAGE) ligands. The chromatin-bound RAGE ligand S100a4 activates RAGE receptors in neighboring surviving tumor cells, leading to Erk activation. In addition, we identified nuclear expulsion products in human patients with breast, bladder and lung cancer and a nuclear expulsion signature correlated with poor prognosis. Collectively, our study demonstrates how apoptotic cell death can enhance the metastatic outgrowth of neighboring live tumor cells.

Schizophrenia and neurosurgery: systematic review and theories.

OBJECTIVE: Despite its relatively low prevalence, schizophrenia has a high burden of illness due to its lifelong effects and the fact that it is often refractory to psychotropic treatment. This review investigated how neurosurgical interventions, primarily neuromodulation through deep brain stimulation (DBS), can mitigate treatment-refractory schizophrenia. Pathophysiological data and ongoing clinical trials were reviewed to suggest which targets hold promise for neurosurgical efficacy. METHODS: A systematic review of the literature was conducted via an electronic search of the PubMed, Scopus, and Web of Science databases. Included papers were human or animal studies of neurosurgical interventions for schizophrenia conducted between 2012 and 2022. An electronic search of ClinicalTrials.gov and the International Clinical Trials Registry Platform was conducted to find ongoing clinical trials. The ROBINS-I (Risk of Bias in Nonrandomized Studies of Interventions) assessment tool was used to evaluate risk of bias in the study. RESULTS: Eight human and 2 rat studies were included in the review. Of the human studies, 5 used DBS targeting the nucleus accumbens, subgenual anterior cingulate cortex, habenula, and substantial nigra pars reticulata. The remaining 3 human studies reported the results of subcaudate tractotomies and anterior capsulotomies. The rat studies investigated DBS of the nucleus accumbens and medial prefrontal cortex. Overall, human studies demonstrated long-term reduction in Positive and Negative Syndrome Scale scores in many participants, with a low incidence of surgical and psychological side effects. The rat studies demonstrated improved prepulse and latent inhibition in the targeted areas after DBS. CONCLUSIONS: As identified in this review, recent studies have investigated the potential effects of therapeutic DBS for schizophrenia, with varying results. DBS targets that have been explored include the hippocampus, subgenual anterior cingulate cortex, habenula, substantia nigra pars reticulata, and medial prefrontal cortex. In addition to DBS, other neuromodulatory techniques such as neuroablation have been studied. Current evidence suggests that neuroablation in the subcaudate tract and anterior capsulotomy may be beneficial for some patients. The authors recommend further exploration of neuromodulation for treatment-refractory schizophrenia, under the condition that rigorous standards be upheld when considering surgical candidacy for these treatments, given that their safety and efficacy remain to be determined.

Dysregulation of Mitochondrial Translation Caused by CBFB Deficiency Cooperates with Mutant PIK3CA and Is a Vulnerability in Breast Cancer.

Understanding functional interactions between cancer mutations is an attractive strategy for discovering unappreciated cancer pathways and developing new combination therapies to improve personalized treatment. However, distinguishing driver gene pairs from passenger pairs remains challenging. Here, we designed an integrated omics approach to identify driver gene pairs by leveraging genetic interaction analyses of top mutated breast cancer genes and the proteomics interactome data of their encoded proteins. This approach identified that PIK3CA oncogenic gain-of-function (GOF) and CBFB loss-of-function (LOF) mutations cooperate to promote breast tumor progression in both mice and humans. The transcription factor CBFB localized to mitochondria and moonlighted in translating the mitochondrial genome. Mechanistically, CBFB enhanced the binding of mitochondrial mRNAs to TUFM, a mitochondrial translation elongation factor. Independent of mutant PI3K, mitochondrial translation defects caused by CBFB LOF led to multiple metabolic reprogramming events, including defective oxidative phosphorylation, the Warburg effect, and autophagy/mitophagy addiction. Furthermore, autophagy and PI3K inhibitors synergistically killed breast cancer cells and impaired the growth of breast tumors, including patient-derived xenografts carrying CBFB LOF and PIK3CA GOF mutations. Thus, our study offers mechanistic insights into the functional interaction between mutant PI3K and mitochondrial translation dysregulation in breast cancer progression and provides a strong preclinical rationale for combining autophagy and PI3K inhibitors in precision medicine for breast cancer. SIGNIFICANCE: CBFB-regulated mitochondrial translation is a regulatory step in breast cancer metabolism and synergizes with mutant PI3K in breast cancer progression.

Secretoglobin 3A2 protects lung from developing cigarette smoke-induced pulmonary emphysema.

Secretoglobin (SCGB) 3A2 is a bioactive molecule exhibiting various functions such as improving allergic airway inflammation and pulmonary fibrosis and promoting bronchial branching and proliferation during lung development. To determine if and how SCGB3A2 is involved in chronic obstructive pulmonary disease (COPD), a multifactorial disease with both airway and emphysematous lesions, a COPD mouse model was created by exposing Scgb3a2-deficient (KO), Scgb3a2-lung-specific overexpressing (TG), and wild type (WT) mice to cigarette smoke (CS) for 6 months. The KO mice showed loss of lung structure under control condition, and CS exposure resulted in more expansion of airspace and destruction of alveolar wall than WT mouse lungs. In contrast, TG mouse lungs showed no significant changes after CS exposure. SCGB3A2 increased the expression and phosphorylation of signal transducers and activators of transcription (STAT)1 and STAT3, and the expression of α1-antitrypsin (A1AT) in mouse lung fibroblast-derived MLg cells and mouse lung epithelial-derived MLE-15 cells. In MLg cells, A1AT expression was decreased in Stat3-knockdown cells, and increased upon Stat3 overexpression. STAT3 formed a homodimer when cells were stimulated with SCGB3A2. Chromatin immunoprecipitation and reporter assays demonstrated that STAT3 binds to specific binding sites on the Serpina1a gene encoding A1AT and upregulates its transcription in lung tissues of mice. Furthermore, nuclear localization of phosphorylated STAT3 upon SCGB3A2 stimulation was detected by immunocytochemistry. These findings demonstrate that SCGB3A2 protects the lungs from the development of CS-induced emphysema by regulating A1AT expression through STAT3 signaling.

Primary Total Laryngectomy versus Organ Preservation for Locally Advanced T3/T4a Laryngeal Cancer.

OBJECTIVE: Organ preservation (OP) treatment for advanced laryngeal cancer has increased compared to primary total laryngectomy. Our study compares oncologic and functional outcomes between these approaches. STUDY DESIGN: Retrospective cohort study. SETTING: Single tertiary care institution. METHODS: Retrospective review of patients receiving primary total laryngectomy or OP for laryngeal cancer between 1/1/2000 and 12/31/2018. RESULTS: A total of 118 patients received primary total laryngectomy and 119 received OP. Overall survival was similar between total laryngectomy and OP. When stratified by T stage, disease-free survival was worse among T3 patients receiving OP versus total laryngectomy. In T3 patients, 28 OP patients experienced local recurrence (28.9%) compared to 3 total laryngectomy patients (7.1%; p < 0.01). In total, 20 OP patients with local recurrence received salvage surgery. These patients had similar overall survival to patients who underwent initial total laryngectomy (TL). About 14 OP patients with local recurrence did not receive salvage surgery. About 89 (75.4%) TL patients achieved normal diet as compared to 64 (53.8%) OP patients (p < 0.001). In TL patients, 106 (89.8%) received primary or secondary tracheoesophageal-prosthesis, 82 (77.4%) of whom achieved completely understandable speech. CONCLUSIONS: There was no difference in survival by treatment in T4 patients, possibly because of strict patient selection. However, disease-free survival was worse in T3 patients receiving OP, likely due to a high local recurrence rate. Approximately 40% of patients with local recurrence were not eligible for salvage laryngectomy. TL patients had comparable swallowing and speech outcomes with OP patients. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:1122-1131, 2023.

Metastasis suppressor NME1 in exosomes or liposomes conveys motility and migration inhibition in breast cancer model systems.

Tumor-derived exosomes have documented roles in accelerating the initiation and outgrowth of metastases, as well as in therapy resistance. Little information supports the converse, that exosomes or similar vesicles can suppress metastasis. We investigated the NME1 (Nm23-H1) metastasis suppressor as a candidate for metastasis suppression by extracellular vesicles. Exosomes derived from two cancer cell lines (MDA-MB-231T and MDA-MB-435), when transfected with the NME1 (Nm23-H1) metastasis suppressor, secreted exosomes with NME1 as the predominant constituent. These exosomes entered recipient tumor cells, altered their endocytic patterns in agreement with NME1 function, and suppressed in vitro tumor cell motility and migration compared to exosomes from control transfectants. Proteomic analysis of exosomes revealed multiple differentially expressed proteins that could exert biological functions. Therefore, we also prepared and investigated liposomes, empty or containing partially purified rNME1. rNME1 containing liposomes recapitulated the effects of exosomes from NME1 transfectants in vitro. In an experimental lung metastasis assay the median lung metastases per histologic section was 158 using control liposomes and 15 in the rNME1 liposome group, 90.5% lower than the control liposome group (P = 0.016). The data expand the exosome/liposome field to include metastasis suppressive functions and describe a new translational approach to prevent metastasis.

DNA Methyltransferase 3B-Mediated Intratumoral Heterogeneity and Therapeutic Targeting in Breast Cancer Recurrence and Metastasis.

The mechanisms of how cancer cells are selected and evolve to establish distant metastatic colonies remain unclear. Tumor heterogeneity and lack of biomarkers are some of the most difficult challenges in cancer biology and treatment. Here using mouse models for triple-negative breast cancer (TNBC) metastasis, we report heterogeneous expression of DNA methyltransferase 3B (DNMT3B) in both mouse and human primary tumors. High levels of DNMT3B were correlated with poor clinical outcomes in multiple human breast cancer datasets. Mechanistically, clonal cells with high DNMT3B (DNMT3BH) showed higher vimentin (VIM) expression and displayed enhanced epithelial-to-mesenchymal transition capacity. Deletion of VIM diminished the metastatic phenotype of DNMT3BH cells. Importantly, in preclinical mouse models in which the primary tumors were surgically removed, perioperative targeting of DNMT3B in combination with chemotherapy markedly suppressed tumor recurrence and metastasis. Our studies identify DNMT3B-mediated transcription regulation as an important mediator of tumor heterogeneity and show that DNMT3B is critical for tumor invasion and metastasis, reinforcing its potential as a target for treating metastatic disease. IMPLICATIONS: Our findings of transcriptome changes mediated by DNMT3B provide new mechanistic insight for intratumor heterogeneity and chemoresistance, and therapeutic targeting of DNMT3B in combination with chemotherapy offer additional treatment options for metastatic disease especially for patients with TNBC.

The oxidoreductase CLIC4 is required to maintain mitochondrial function and resistance to exogenous oxidants in breast cancer cells.

The chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H2O2 as a ROS-inducing agent. In intact cells, H2O2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H2O2-induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H2O2-treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells.

Prognostic value of computed tomography scan detection of cartilage invasion in advanced laryngeal cancer treated with primary total laryngectomy.

BACKGROUND: We sought to determine whether detection of cartilage invasion (CI) by computed tomography predicts oncologic outcomes after primary total laryngectomy. METHODS: Retrospective cohort study comparing oncologic outcomes between radiologic versus pathologic diagnosis. RESULTS: Assessment of clear CI versus gestalt CI resulted in 84% versus 48% specificity, 90.9% versus 80.3% positive predictive value (PPV), 60.6% versus 80.3% sensitivity, 44.7% versus 48% negative predictive value (NPV), respectively. Disease-free survival (DFS) was similar between cT4a and cT3/cT2 patients (p = 0.87). DFS trended towards superiority among pT3/pT2 versus pT4a patients (p = 0.18). DFS was similar among patients with CI on radiologist gestalt versus no CI (p = 0.94). Histologically confirmed CI was associated with a hazard ratio (HR) of 1.46 (p = 0.27), gestalt CI 1.13 (p = 0.70), and clear CI 1.61 (p = 0.10) for DFS. CONCLUSION: Gestalt determination of CI results in high sensitivity but low specificity, while clear determination of CI results in moderate sensitivity and high specificity.

Removal of KCNQ2 from parvalbumin-expressing interneurons improves anti-seizure efficacy of retigabine.

Anti-seizure drug (ASD) targets are widely expressed in both excitatory and inhibitory neurons. It remains unknown if the action of an ASD upon inhibitory neurons could counteract its beneficial effects on excitatory neurons (or vice versa), thereby reducing the efficacy of the ASD. Here, we examine whether the efficacy of the ASD retigabine (RTG) is altered after removal of the Kv7 potassium channel subunit KCNQ2, one of its drug targets, from parvalbumin-expressing interneurons (PV-INs). Parvalbumin-Cre (PV-Cre) mice were crossed with Kcnq2-floxed (Kcnq2fl/fl) mice to conditionally delete Kcnq2 from PV-INs. In these conditional knockout mice (cKO, PV-Kcnq2fl/fl), RTG (10 mg/kg, i.p.) significantly delayed the onset of either picrotoxin (PTX, 10 mg/kg, i.p)- or kainic acid (KA, 30 mg/kg, i.p.)-induced convulsive seizures compared to vehicle, while RTG was not effective in wild-type littermates (WT). Immunostaining for KCNQ2 and KCNQ3 revealed that both subunits were enriched at axon initial segments (AISs) of hippocampal CA1 PV-INs, and their specific expression was selectively abolished in cKO mice. Accordingly, the M-currents recorded from CA1 PV-INs and their sensitivity to RTG were significantly reduced in cKO mice. While the ability of RTG to suppress CA1 excitatory neurons in hippocampal slices was unchanged in cKO mice, its suppressive effect on the spike activity of CA1 PV-INs was significantly reduced compared with WT mice. In addition, the RTG-induced suppression on intrinsic membrane excitability of PV-INs in WT mice was significantly reduced in cKO mice. These findings suggest that preventing RTG from suppressing PV-INs improves its anticonvulsant effect.