A PERK-Specific Inhibitor Blocks Metastatic Progression by Limiting Integrated Stress Response-Dependent Survival of Quiescent Cancer Cells.

PURPOSE: The integrated stress response (ISR) kinase PERK serves as a survival factor for both proliferative and dormant cancer cells. We aim to validate PERK inhibition as a new strategy to specifically eliminate solitary disseminated cancer cells (DCC) in secondary sites that eventually reawake and originate metastasis. EXPERIMENTAL DESIGN: A novel clinical-grade PERK inhibitor (HC4) was tested in mouse syngeneic and PDX models that present quiescent/dormant DCCs or growth-arrested cancer cells in micro-metastatic lesions that upregulate ISR. RESULTS: HC4 significantly blocks metastasis, by killing quiescent/slow-cycling ISRhigh, but not proliferative ISRlow DCCs. HC4 blocked expansion of established micro-metastasis that contained ISRhigh slow-cycling cells. Single-cell gene expression profiling and imaging revealed that a significant proportion of solitary DCCs in lungs were indeed dormant and displayed an unresolved ER stress as revealed by high expression of a PERK-regulated signature. In human breast cancer metastasis biopsies, GADD34 expression (PERK-regulated gene) and quiescence were positively correlated. HC4 effectively eradicated dormant bone marrow DCCs, which usually persist after rounds of therapies. Importantly, treatment with CDK4/6 inhibitors (to force a quiescent state) followed by HC4 further reduced metastatic burden. In HNSCC and HER2+ cancers HC4 caused cell death in dormant DCCs. In HER2+ tumors, PERK inhibition caused killing by reducing HER2 activity because of sub-optimal HER2 trafficking and phosphorylation in response to EGF. CONCLUSIONS: Our data identify PERK as a unique vulnerability in quiescent or slow-cycling ISRhigh DCCs. The use of PERK inhibitors may allow targeting of pre-existing or therapy-induced growth arrested "persister" cells that escape anti-proliferative therapies.

Pancreas Rejection in the Artificial Intelligence Era: New Tool for Signal Patients at Risk.

INTRODUCTION: Pancreas transplantation is currently the only treatment that can re-establish normal endocrine pancreatic function. Despite all efforts, pancreas allograft survival and rejection remain major clinical problems. The purpose of this study was to identify features that could signal patients at risk of pancreas allograft rejection. METHODS: We collected 74 features from 79 patients who underwent simultaneous pancreas-kidney transplantation (SPK) and used two widely-applicable classification methods, the Naive Bayesian Classifier and Support Vector Machine, to build predictive models. We used the area under the receiver operating characteristic curve and classification accuracy to evaluate the predictive performance via leave-one-out cross-validation. RESULTS: Rejection events were identified in 13 SPK patients (17.8%). In feature selection approach, it was possible to identify 10 features, namely: previous treatment for diabetes mellitus with long-term Insulin (U/I/day), type of dialysis (peritoneal dialysis, hemodialysis, or pre-emptive), de novo DSA, vPRA_Pre-Transplant (%), donor blood glucose, pancreas donor risk index (pDRI), recipient height, dialysis time (days), warm ischemia (minutes), recipient of intensive care (days). The results showed that the Naive Bayes and Support Vector Machine classifiers prediction performed very well, with an AUROC and classification accuracy of 0.97 and 0.87, respectively, in the first model and 0.96 and 0.94 in the second model. CONCLUSION: Our results indicated that it is feasible to develop successful classifiers for the prediction of graft rejection. The Naive Bayesian generated nomogram can be used for rejection probability prediction, thus supporting clinical decision making.

5-Azacytidine- and retinoic-acid-induced reprogramming of DCCs into dormancy suppresses metastasis via restored TGF-ß-SMAD4 signaling.

Disseminated cancer cells (DCCs) in secondary organs can remain dormant for years to decades before reactivating into overt metastasis. Microenvironmental signals leading to cancer cell chromatin remodeling and transcriptional reprogramming appear to control onset and escape from dormancy. Here, we reveal that the therapeutic combination of the DNA methylation inhibitor 5-azacytidine (AZA) and the retinoic acid receptor ligands all-trans retinoic acid (atRA) or AM80, an RARα-specific agonist, promotes stable dormancy in cancer cells. Treatment of head and neck squamous cell carcinoma (HNSCC) or breast cancer cells with AZA+atRA induces a SMAD2/3/4-dependent transcriptional program that restores transforming growth factor ß (TGF-ß)-signaling and anti-proliferative function. Significantly, either combination, AZA+atRA or AZA+AM80, strongly suppresses HNSCC lung metastasis formation by inducing and maintaining solitary DCCs in a SMAD4+/NR2F1+ non-proliferative state. Notably, SMAD4 knockdown is sufficient to drive resistance to AZA+atRA-induced dormancy. We conclude that therapeutic doses of AZA and RAR agonists may induce and/or maintain dormancy and significantly limit metastasis development.

A Collaborative Approach for the Development of a Standardized Set of Patient-Centered Outcomes in Head and Neck Cancers.

INTRODUCTION: Head and neck cancers remain a significant health burden worldwide. Standardizing the care provided to these patients through the systematic measurement of established indicators is key to improve their outcomes. The aim of this study was to establish a relevant set of outcome indicators in this condition and identify measurement tools and requirements to do so. MATERIAL AND METHODS: One scientific committee and two regional working groups worked in a stepwise manner to narrow down an initial list of potential outcome indicators retrieved from an exhaustive literature review to a smaller set of outcome indicators according to their clinical practice. This was assessed by one representative of a head and neck cancer patient association until a final set of indicators was reached. RESULTS: A total of 164 outcome indicators comprising case-mix, outcomes, and adverse events dimensions were retrieved from the literature. These were reduced to a working set of 79 outcome indicators by the Scientific Committee and divided into seven categories including demographics, clinical status, tumor-related parameters, nutritional status, treatment, health and quality of life parameters and survival. Subsequently, these indicators were further reduced to a set of 50 indicators by the regional working groups and to a set of 49 indicators by the final Scientific Committee assessment. Finally, the discussed indicators were appraised by a head and neck cancer patient association, which added the 'rehabilitation' category, a key parameter to these patients. CONCLUSION: An initial set of outcome indicators for head and neck cancer was systematically developed aiming to standardize the care provided to these patients across institutions at national level and identify measurement tools and requirements to measure those indicators. This standard set should be continuously improved and consistently adopted in the different clinical and national settings.

ZFP281 drives a mesenchymal-like dormancy program in early disseminated breast cancer cells that prevents metastatic outgrowth in the lung.

Increasing evidence shows that cancer cells can disseminate from early evolved primary lesions much earlier than the classical metastasis models predicted. Here, we reveal at a single-cell resolution that mesenchymal-like (M-like) and pluripotency-like programs coordinate dissemination and a long-lived dormancy program of early disseminated cancer cells (DCCs). The transcription factor ZFP281 induces a permissive state for heterogeneous M-like transcriptional programs, which associate with a dormancy signature and phenotype in vivo. Downregulation of ZFP281 leads to a loss of an invasive, M-like dormancy phenotype and a switch to lung metastatic outgrowth. We also show that FGF2 and TWIST1 induce ZFP281 expression to induce the M-like state, which is linked to CDH1 downregulation and upregulation of CDH11. We found that ZFP281 not only controls the early dissemination of cancer cells but also locks early DCCs in a dormant state by preventing the acquisition of an epithelial-like proliferative program and consequent metastases outgrowth.

Insights from the use of erythropoietin in experimental Chagas disease.

In addition to the long-established role in erythropoiesis, erythropoietin (Epo) has protective functions in a variety of tissues, including the heart. This is the most affected organ in chronic Chagas disease, caused by the protozoan Trypanosoma cruzi. Despite seven million people being infected with T. cruzi worldwide, there is no effective treatment preventing the disease progression to the chronic phase when the pathological involvement of the heart is often observed. Chronic chagasic cardiomyopathy has a wide variety of manifestations, like left ventricular systolic dysfunction, dilated cardiomyopathy, and heart failure. Since Epo may help maintain cardiac function by reducing myocardial necrosis, inflammation, and fibrosis, this study aimed to evaluate whether the Epo has positive effects on experimental Chagas disease. For that, we assessed the earlier (acute phase) and also the later (chronic phase) use of Epo in infected C57BL/6 mice. Blood cell count, biochemical parameters, parasitic load, and echocardiography data were evaluated. In addition, histopathological analysis was carried out. Our data showed that Epo had no trypanocide effect nor did it modify the production of anti-T. cruzi antibodies. Epo-treated groups exhibited parasitic burden much lower in the heart compared to blood. No pattern of hematological changes was observed combining infection with treatment with Epo. Chronic Epo administration reduced CK-MB serum activity from d0 to d180, irrespectively of T. cruzi infection. Likewise, echocardiography and histological results indicate that Epo treatment is more effective in the chronic phase of experimental Chagas disease. Since treatment is one of the greatest challenges of Chagas disease, alternative therapies should be investigated, including Epo combined with benznidazole.

A tumor-derived type III collagen-rich ECM niche regulates tumor cell dormancy.

Cancer cells disseminate and seed in distant organs, where they can remain dormant for many years before forming clinically detectable metastases. Here we studied how disseminated tumor cells sense and remodel the extracellular matrix (ECM) to sustain dormancy. ECM proteomics revealed that dormant cancer cells assemble a type III collagen-enriched ECM niche. Tumor-derived type III collagen is required to sustain tumor dormancy, as its disruption restores tumor cell proliferation through DDR1-mediated STAT1 signaling. Second-harmonic generation two-photon microscopy further revealed that the dormancy-to-reactivation transition is accompanied by changes in type III collagen architecture and abundance. Analysis of clinical samples revealed that type III collagen levels were increased in tumors from patients with lymph node-negative head and neck squamous cell carcinoma compared to patients who were positive for lymph node colonization. Our data support the idea that the manipulation of these mechanisms could serve as a barrier to metastasis through disseminated tumor cell dormancy induction.

An NR2F1-specific agonist suppresses metastasis by inducing cancer cell dormancy.

We describe the discovery of an agonist of the nuclear receptor NR2F1 that specifically activates dormancy programs in malignant cells. The agonist led to a self-regulated increase in NR2F1 mRNA and protein and downstream transcription of a novel dormancy program. This program led to growth arrest of an HNSCC PDX line, human cell lines, and patient-derived organoids in 3D cultures and in vivo. This effect was lost when NR2F1 was knocked out by CRISPR-Cas9. RNA sequencing revealed that agonist treatment induces transcriptional changes associated with inhibition of cell cycle progression and mTOR signaling, metastasis suppression, and induction of a neural crest lineage program. In mice, agonist treatment resulted in inhibition of lung HNSCC metastasis, even after cessation of the treatment, where disseminated tumor cells displayed an NR2F1hi/p27hi/Ki-67lo/p-S6lo phenotype and remained in a dormant single-cell state. Our work provides proof of principle supporting the use of NR2F1 agonists to induce dormancy as a therapeutic strategy to prevent metastasis.

Integration of segmented regression analysis with weighted gene correlation network analysis identifies genes whose expression is remodeled throughout physiological aging in mouse tissues.

Gene expression alterations occurring with aging have been described for a multitude of species, organs, and cell types. However, most of the underlying studies rely on static comparisons of mean gene expression levels between age groups and do not account for the dynamics of gene expression throughout the lifespan. These studies also tend to disregard the pairwise relationships between gene expression profiles, which may underlie commonly altered pathways and regulatory mechanisms with age. To overcome these limitations, we have combined segmented regression analysis with weighted gene correlation network analysis (WGCNA) to identify high-confidence signatures of aging in the brain, heart, liver, skeletal muscle, and pancreas of C57BL/6 mice in a publicly available RNA-Seq dataset (GSE132040). Functional enrichment analysis of the overlap of genes identified in both approaches showed that immune- and inflammation-related responses are prominently altered in the brain and the liver, while in the heart and the muscle, aging affects amino and fatty acid metabolism, and tissue regeneration, respectively, which reflects an age-related global loss of tissue function. We also explored sexual dimorphism in the aging mouse transcriptome and found the liver and the muscle to have the most pronounced gender differences in gene expression throughout the lifespan, particularly in proteostasis-related pathways. While the data showed little overlap among the age-dysregulated genes between tissues, aging triggered common biological processes in distinct tissues, which we highlight as important features of murine tissue physiological aging.

Bone marrow NG2+/Nestin+ mesenchymal stem cells drive DTC dormancy via TGFß2.

In the bone marrow (BM) microenvironment, where breast cancer (BC) disseminated tumour cells (DTCs) can remain dormant for decades, NG2+/Nestin+ mesenchymal stem cells (MSCs) promote hematopoietic stem cell quiescence. Here, we reveal that periarteriolar BM-resident NG2+/Nestin+ MSCs can also instruct BC DTCs to enter dormancy. NG2+/Nestin+ MSCs produce TGFß2 and BMP7 and activate a quiescence pathway dependent on TGFBRIII and BMPRII, which via p38-kinase result in p27 induction. Genetic depletion of MSCs or conditional knock-out of TGFß2 in MSCs using an NG2-CreER driver led to bone metastatic outgrowth of otherwise dormant p27+/Ki67- DTCs. Also ER+ BC patients without systemic recurrence displayed higher frequency of TGFß2 and BMP7 detection in the BM. Our results provide a direct proof that HSC dormancy niches control BC DTC dormancy and suggest that aging or extrinsic factors that affect the NG2+/Nestin+ MSC niche homeostasis may result in a break from dormancy and BC bone relapse.

Surgical Closure of Tracheoesophageal Puncture Without a Flap Interposition.

Tracheoesophageal puncture for voice prosthesis placement is often used in vocal rehabilitation of patients undergoing total laryngectomy. Although its closure can occur spontaneously, some patients require a surgical procedure. We propose a surgical technique, without flap interposition, that begins with careful separation of the esophagus and trachea and identification of the site of tracheoesophageal fistula. After continuous suture closure of the esophagus, the anterior segment of the first tracheal rings is vertically incised to facilitate tracheal closure in a suture without tension. Finally, a small pectoral skin flap is made and mobilized to suture to the free edges of the sectioned tracheal rings, thus reducing the risk of tracheal stenosis. Four patients underwent this procedure with uneventful postoperative evolution and permanent closure of the fistula.

The current paradigm and challenges ahead for the dormancy of disseminated tumor cells.

Disseminated tumor cells (DTCs) are known to enter a state of dormancy that is achieved via growth arrest of DTCs and/or a form of population equilibrium state, strongly influenced by the organ microenvironment. During this time, expansion of residual disseminated cancer is paused and DTCs survive to fuel relapse, sometimes decades later. This notion has opened a new window of opportunity for intervening and preventing relapse. Here we review recent data that have further augmented the understanding of cancer dormancy and discuss how this is leading to new strategies for monitoring and targeting dormant cancer.

SRC inhibition prevents P-cadherin mediated signaling and function in basal-like breast cancer cells.

BACKGROUND: Basal-like breast cancer (BLBC) is a poor prognosis subgroup of triple-negative carcinomas that still lack specific target therapies and accurate biomarkers for treatment selection. P-cadherin is frequently overexpressed in these tumors, promoting cell invasion, stem cell activity and tumorigenesis by the activation of Src-Family kinase (SRC) signaling. Therefore, our aim was to evaluate if the treatment of BLBC cells with dasatinib, the FDA approved SRC inhibitor, would impact on P-cadherin induced tumor aggressive behavior. METHODS: P-cadherin and SRC expression was evaluated in a series of invasive Breast Cancer and contingency tables and chi-square tests were performed. Cell-cell adhesion measurements were performed by Atomic Force Microscopy, where frequency histograms and Gaussian curves were applied. 2D and 3D cell migration and invasion, proteases secretion and self-renew potential were evaluated in vitro. Student's t-tests were used to determine statistically significant differences. The cadherin/catenin complex interactions were evaluated by in situ proximity-ligation assay, and statistically significant results were determined by using Mann-Whitney test with a Bonferroni correction. In vivo xenograft mouse models were used to evaluate the impact of dasatinib on tumor growth and survival. ANOVA test was used to evaluate the differences in tumor size, considering a confidence interval of 95%. Survival curves were estimated by the Kaplan-Meier's method, using the log-rank test to assess significant differences for mice overall survival. RESULTS: Our data demonstrated that P-cadherin overexpression is significantly associated with SRC activation in breast cancer cells, which was also validated in a large series of primary tumor samples. SRC activity suppression with dasatinib significantly prevented the in vitro functional effects of P-cadherin overexpressing cells, as well as their in vivo tumorigenic and metastatic ability, by increasing mice overall survival. Mechanistically, SRC inhibition affects P-cadherin downstream signaling, rescues the E-cadherin/p120-catenin complex to the cell membrane, recovering cell-cell adhesion function. CONCLUSIONS: In conclusion our findings show that targeting P-cadherin/SRC signaling and functional activity may open novel therapeutic opportunities for highly aggressive and poor prognostic basal-like breast cancer.

The Different Routes to Metastasis via Hypoxia-Regulated Programs.

Hypoxia is linked to metastasis; however, how it affects metastatic progression is not clear due to limited consensus in the literature. We posit that this lack of consensus is due to hypoxia being studied using different approaches, such as in vitro, primary tumor, or metastasis assays in an isolated manner. Here, we review the pros and cons of in vitro hypoxia assays, highlight in vivo studies that inform on physiological hypoxia, and review the evidence that primary tumor hypoxia might influence the fate of disseminated tumor cells (DTCs) in secondary organs. Our analysis suggests that consensus can be reached by using in vivo methods of study, which also allow better modeling of how hypoxia affects DTC fate and metastasis.

Phenotypic heterogeneity of disseminated tumour cells is preset by primary tumour hypoxic microenvironments.

Hypoxia is a poor-prognosis microenvironmental hallmark of solid tumours, but it is unclear how it influences the fate of disseminated tumour cells (DTCs) in target organs. Here we report that hypoxic HNSCC and breast primary tumour microenvironments displayed upregulation of key dormancy (NR2F1, DEC2, p27) and hypoxia (GLUT1, HIF1α) genes. Analysis of solitary DTCs in PDX and transgenic mice revealed that post-hypoxic DTCs were frequently NR2F1hi/DEC2hi/p27hi/TGFß2hi and dormant. NR2F1 and HIF1α were required for p27 induction in post-hypoxic dormant DTCs, but these DTCs did not display GLUT1hi expression. Post-hypoxic DTCs evaded chemotherapy and, unlike ER- breast cancer cells, post-hypoxic ER+ breast cancer cells were more prone to enter NR2F1-dependent dormancy. We propose that primary tumour hypoxic microenvironments give rise to a subpopulation of dormant DTCs that evade therapy. These post-hypoxic dormant DTCs may be the source of disease relapse and poor prognosis associated with hypoxia.

Axl is required for TGF-ß2-induced dormancy of prostate cancer cells in the bone marrow.

Disseminated prostate cancer (PCa) cells in the marrow survive for years without evidence of proliferation, while maintaining the capacity to develop into metastatic lesions. These dormant disseminated tumor cells (DTCs) may reside in close proximity to osteoblasts, while expressing high levels of Axl, one of the tyrosine kinase receptors for growth arrest specific 6 (Gas6). Yet how Axl regulates DTC proliferation in marrow remains undefined. Here, we explored the impact of the loss of Axl in PCa cells (PC3 and DU145) on the induction of their dormancy when they are co-cultured with a pre-osteoblastic cell line, MC3T3-E1. MC3T3-E1 cells dramatically decrease the proliferation of PCa cells, however this suppressive effect of osteoblasts is significantly reduced by the reduction of Axl expression in PCa cells. Interestingly, expression of both TGF-ß and its receptors were regulated by Axl expression in PCa cells, while specific blockade of TGF-ß signaling limited the ability of the osteoblasts to induce dormancy of PCa cells. Finally, we found that both Gas6 and Axl are required for TGF-ß2-mediated cell growth suppression. Taken together, these data suggest that a loop between the Gas6/Axl axis and TGF-ß2 signaling plays a significant role in the induction of PCa cell dormancy.

Volatile profile of cashew apple juice fibers from different production steps.

This study aimed to determine the volatile profile of cashew apple fibers to verify which compounds are still present after successive washings and thus might be responsible for the undesirable remaining cashew-like aroma present in this co-product, which is used to formulate food products like vegetarian burgers and cereal bars. Fibers were obtained from cashew apple juice processing and washed five times in an expeller press. Compounds were analyzed by the headspace solid-phase micro extraction technique (HS-SPME) and gas chromatography-mass spectrometry (GC-MS), using a DB-5 column. Sensory analysis was also performed to compare the intensity of the cashew-like aroma of the fibers with the original juice. Altogether, 80 compounds were detected, being esters and terpenes the major chemical classes. Among the identified substances, 14 were classified as odoriferous in the literature, constituting the matrix used in the Principal Component Analysis (PCA). Odoriferous esters were substantially reduced, but many compounds were extracted by the strength used in the expeller press and remained until the last wash. Among them are the odoriferous compounds ethyl octanoate, γ-dodecalactone, (E)-2-decenal, copaene, and caryophyllene that may contribute for the mild but still perceptible cashew apple aroma in the fibers that have been pressed and washed five times. Development of a deodorization process should include reduction of pressing force and stop at the second wash, to save water and energy, thus reducing operational costs and contributing to process sustainability.

A unique glyceryl diglycoside identified in the thermophilic, radiation-resistant bacterium Rubrobacter xylanophilus.

The solute pool of the actinobacterium Rubrobacter xylanophilus has been investigated as a function of the growth temperature and concentration of NaCl in the medium (Empadinhas et al. Extremophiles 11: 667-673, 2007). Changing the carbon source from glucose to maltose in a minimal growth medium led to the accumulation of an unknown organic compound whose structure was investigated by NMR and confirmed by chemical synthesis in the present study as: (2R)-2-(1-O-α-D-mannopyranosyl)-3-(1-O-α-D-glucopyranosyl)-D-glycerate (MGlyG). In addition to this newly identified diglycoside, the solute pool of R. xylanophilus included trehalose, mannosylglycerate, di-myo-inositol phosphate and di-N-acetyl-glucosamine phosphate. The structure of MGlyG was established by NMR and confirmed by chemical synthesis. The availability of g-amounts of the synthetic material allowed us to perform stabilization tests on three model enzymes (malate dehydrogenase, staphylococcal nuclease, and lysozyme), and compare the efficacy of MGlyG with other natural glyceryl glycosides, such as α-D-mannosyl-D-glycerate, α-D-glucosyl-D-glycerate and α-D-glucosyl-(1 → 6)-α-D-glucosyl-(1 → 2)-D-glycerate.

The basal epithelial marker P-cadherin associates with breast cancer cell populations harboring a glycolytic and acid-resistant phenotype.

BACKGROUND: Cancer stem cells are hypoxia-resistant and present a preponderant glycolytic metabolism. These characteristics are also found in basal-like breast carcinomas (BLBC), which show increased expression of cancer stem cell markers.Recently, we demonstrated that P-cadherin, a biomarker of BLBC and a poor prognostic factor in this disease, mediates stem-like properties and resistance to radiation therapy. Thus, the aim of the present study was to evaluate if P-cadherin expression was associated to breast cancer cell populations with an adapted phenotype to hypoxia. METHODS: Immunohistochemistry was performed to address the expression of P-cadherin, hypoxic, glycolytic and acid-resistance biomarkers in primary human breast carcinomas. In vitro studies were performed using basal-like breast cancer cell lines. qRT-PCR, FACS analysis, western blotting and confocal microscopy were used to assess the expression of P-cadherin after HIF-1α stabilization, achieved by CoCl2 treatment. siRNA-mediated knockdown was used to silence the expression of several targets and qRT-PCR was employed to evaluate the effects of P-cadherin on HIF-1α signaling. P-cadherin high and low breast cancer cell populations were sorted by FACS and levels of GLUT1 and CAIX were assessed by FACS and western blotting. Mammosphere forming efficiency was used to determine the stem cell activity after specific siRNA-mediated knockdown, further confirmed by western blotting. RESULTS: We demonstrated that P-cadherin overexpression was significantly associated with the expression of HIF-1α, GLUT1, CAIX, MCT1 and CD147 in human breast carcinomas. In vitro, we showed that HIF-1α stabilization was accompanied by increased membrane expression of P-cadherin and that P-cadherin silencing led to a decrease of the mRNA levels of GLUT1 and CAIX. We also found that the cell fractions harboring high levels of P-cadherin were the same exhibiting more GLUT1 and CAIX expression. Finally, we showed that P-cadherin silencing significantly decreases the mammosphere forming efficiency in the same range as the silencing of HIF-1α, CAIX or GLUT1, validating that all these markers are being expressed by the same breast cancer stem cell population. CONCLUSIONS: Our results establish a link between aberrant P-cadherin expression and hypoxic, glycolytic and acid-resistant breast cancer cells, suggesting a possible role for this marker in cancer cell metabolism.