CAV1 inhibits metastatic potential in melanomas through suppression of the Integrin/Src/FAK signaling pathway.

Caveolin-1 (CAV1) is the main structural component of Caveolae which are plasma membrane invaginations that participate in vesicular trafficking and signal transduction events. Although, evidence has recently accumulated describing the function of CAV1 in several cancer types, its role in melanoma tumor formation and progression remains poorly explored. Here, by employing B16F10 melanoma cells as an experimental system, we directly explore the function of CAV1 in melanoma tumor growth and metastasis. We first show that CAV1 expression promotes proliferation while it suppresses migration and invasion of B16F10 cells in vitro. When orthotopically implanted in the skin of mice, B16F10 cells expressing CAV1 form tumors that are similar in size to their control counterpart. An experimental metastasis assay demonstrates that CAV1 expression suppresses the ability of B16F10 cells to form lung metastases in C57Bl/6 syngeneic mice. Additionally, CAV1 protein and mRNA levels are found to be significantly reduced in human metastatic melanoma cell lines and human tissue from metastatic lesions. Finally, we demonstrate that following integrin activation, B16F10 cells expressing CAV1 display reduced expression levels and activity of FAK and Src proteins. CAV1 expression also markedly reduces the expression levels of beta3 Integrin in B16F10 melanoma cells. In summary, our findings provide experimental evidence that CAV1 may function as an antimetastatic gene in malignant melanoma

Metabolic remodeling of the tumor microenvironment: migration stimulating factor (MSF) reprograms myofibroblasts toward lactate production, fueling anabolic tumor growth.

Migration stimulating factor (MSF) is a genetically truncated N-terminal isoform of fibronectin that is highly expressed during mammalian development in fetal fibroblasts, and during tumor formation in human cancer-associated myofibroblasts. However, its potential functional role in regulating tumor metabolism remains unexplored. Here, we generated an immortalized fibroblast cell line that recombinantly overexpresses MSF and studied their properties relative to vector-alone control fibroblasts. Our results indicate that overexpression of MSF is sufficient to confer myofibroblastic differentiation, likely via increased TGF-b signaling. In addition, MSF activates the inflammation-associated transcription factor NFκB, resulting in the onset of autophagy/mitophagy, thereby driving glycolytic metabolism (L-lactate production) in the tumor microenvironment. Consistent with the idea that glycolytic fibroblasts fuel tumor growth (via L-lactate, a high-energy mitochondrial fuel), MSF fibroblasts significantly increased tumor growth, by up to 4-fold. Mechanistic dissection of the MSF signaling pathway indicated that Cdc42 lies downstream of MSF and fibroblast activation. In accordance with this notion, Cdc42 overexpression in immortalized fibroblasts was sufficient to drive myofibroblast differentiation, to provoke a shift towards glycolytic metabolism and to promote tumor growth by up to 2-fold. In conclusion, the MSF/Cdc42/NFκB signaling cascade may be a critical druggable target in preventing Warburg-like cancer metabolism in tumor-associated fibroblasts. Thus, MSF functions in the metabolic remodeling of the tumor microenvironment by metabolically reprogramming cancer-associated fibroblasts toward glycolytic metabolism

Cytokine storm and histopathological findings in 60 cases of COVID-19-related death: from viral load research to immunohistochemical quantification of major players IL-1\u3b2, IL-6, IL-15 and TNF-\u3b1

This study involves the histological analysis of samples taken during autopsies in cases of COVID-19 related death to evaluate the inflammatory cytokine response and the tissue localization of the virus in various organs. In all the selected cases, SARS-CoV-2 RT-PCR on swabs collected from the upper (nasopharynx and oropharynx) and/or the lower respiratory (trachea and primary bronchi) tracts were positive. Tissue localization of SARS-CoV-2 was detected using antibodies against the nucleoprotein and the spike protein. Overall, we tested the hypothesis that the overexpression of proinflammatory cytokines plays an important role in the development of COVID-19-associated pneumonia by estimating the expression of multiple cytokines (IL-1\u3b2, IL-6, IL-10, IL-15, TNF-\u3b1, and MCP-1), inflammatory cells (CD4, CD8, CD20, and CD45), and fibrinogen. Immunohistochemical staining showed that endothelial cells expressed IL-1\u3b2 in lung samples obtained from the COVID-19 group (p\u2009<\u20090.001). Similarly, alveolar capillary endothelial cells showed strong and diffuse immunoreactivity for IL-6 and IL-15 in the COVID-19 group (p\u2009<\u20090.001). TNF-\u3b1 showed a higher immunoreactivity in the COVID-19 group than in the control group (p\u2009<\u20090.001). CD8\u2009+\u2009T cells where more numerous in the lung samples obtained from the COVID-19 group (p\u2009<\u20090.001). Current evidence suggests that a cytokine storm is the major cause of acute respiratory distress syndrome (ARDS) and multiple organ failure and is consistently linked with fatal outcomes

Identification of Novel Genetic Loci Associated with Thyroid Peroxidase Antibodies and Clinical Thyroid Disease

Peer reviewe

Post-coital death in chronic sildenafil abuser

open3noLetterNo abstract availableopenD'Errico, Stefano; Bonuccelli, Diana; Neri, MargheritaD'Errico, Stefano; Bonuccelli, Diana; Neri, Margherit

Is hospital autopsy auditing suitable for clinical risk management? Actualities and perspectives of auditing in the autopsy room following Italian Law 24/2017 on patient safety

BACKGROUND: autopsies are a valuable tool for understanding the physiopathology of a disease and it is the gold standard to assess the cause of death. The clinical autopsy is the ultimate medical service for a patient and plays a crucial role in the context of quality control, education of physicians and other medical personnel, as well as mitigation of risk of malpractice claims.OBJECTIVE: This study aims to demonstrate the importance of improving an autopsy service and the relevance of this investigation procedure in daily clinical practice by evaluating the rate of major discrepancies between the assumed cause of death and the ascertained cause of death after a complete post mortem investigation. A further aim is to classify these discrepancies as class I or class II discrepancies according to Goldman's criteria in order to asses performance quality.METHODS: a retrospective study of the hospital autopsies performed from June 2018 to March 2020 was conducted by considering a diversified dataset, including age and sex of the deceased as well as the clinical and pathological causes of death.RESULTS: 362 cases were taken into consideration. Major discrepancies were found in 71.3% of cases, with a class I error of 22.7% and a class II error of 48.6%. The most frequent misdiagnoses were cardiovascular disorder, embolism and aneurism rupture.DISCUSSION: The rate of major discrepancies and the rate of class I and class II errors are way above the rate found in literature. Despite the high rate of major discrepancy evidences collected from hospital autopsies (i.e. certainty of the cause of death, unknown comorbidities) strengthened the legal defense in cases of medical malpractice litigation. In our experience, by accurately determining the cause of death, revealing new or unexpected findings and possible diagnostic or technical errors, postmortem examinations can significantly contribute to the improvement of team performance and quality of care.CONCLUSION: The presence of clinicians during autopsy and the early sharing of results can be considered a new auditing strategy for hard clinical cases. Finally, by providing a clearer understanding of the nature and cause of the illness, the autopsy results assisted in the grieving process by reassuring family members that action or inaction on their part had not contributed to the death

Activation of Type I and Type II Interferon Signaling in SARS-CoV-2-Positive Thyroid Tissue of Patients Dying from COVID-19

: Background: Thyroid dysfunctions have been reported after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. However, the biological mechanisms behind these conditions remain unexplored. Herein, we report on changes of the immune transcriptome in autoptic thyroid tissues of people who have died from coronavirus disease 2019 (COVID-19). Methods: Twenty-five autoptic thyroid specimens of subjects dying from COVID-19 were investigated. Eleven autoptic thyroid specimens of subjects dying from causes other than infectious conditions served as controls. RNA transcripts of 770 immune-related genes together with RNA genomes of multiple coronavirus types were measured by the nCounter system. Reverse transcription-polymerase chain reaction for two SARS-CoV-2 genes was used to assess virus positivity. Results were validated by immunohistochemistry. Results: The SARS-CoV-2 genome and antigens were detected in 9 of 25 (36%) thyroid specimens from the COVID-19 cohort. Virus-negative thyroid tissues from COVID-19 subject did not show changes of gene transcription nor significant numbers of infiltrating immune cells. Conversely, SARS-CoV-2-positive thyroid specimens showed marked upregulation of immune genes, especially those proper of the type I and type II interferon (IFN) pathways. In infected tissues, infiltrates of innate immune cells (macrophages and polymorphonuclear neutrophils) were prevalent. Conclusions: The thyroid gland can be directly infected by the SARS-CoV-2. Infection strongly activates IFN pathways. The direct viral insult combined with an intense immune response may trigger or worsen thyroid conditions in predisposed individuals

The milk protein α-casein functions as a tumor suppressor via activation of STAT1 signaling, effectively preventing breast cancer tumor growth and metastasis

Here, we identified the milk protein α-casein as a novel suppressor of tumor growth and metastasis. Briefly, Met-1 mammary tumor cells expressing α-casein showed a ~5-fold reduction in tumor growth and a near 10-fold decrease in experimental metastasis. To identify the molecular mechanism(s), we performed genome-wide transcriptional profiling. Interestingly, our results show that α-casein upregulates gene transcripts associated with interferon/STAT1 signaling and downregulates genes associated with “stemness.” These findings were validated by immunoblot and FACS analysis, which showed the upregulation and hyperactivation of STAT1 and a decrease in the number of CD44(+) “cancer stem cells.” These gene signatures were also able to predict clinical outcome in human breast cancer patients. Thus, we conclude that a lactation-based therapeutic strategy using recombinant α-casein would provide a more natural and non-toxic approach to the development of novel anticancer therapies

Matrix remodeling stimulates stromal autophagy, "fueling" cancer cell mitochondrial metabolism and metastasis

We have previously demonstrated that loss of stromal caveolin-1 (Cav-1) in cancer-associated fibroblasts is a strong and independent predictor of poor clinical outcome in human breast cancer patients. However, the signaling mechanism(s) by which Cav-1 downregulation leads to this tumor-promoting microenvironment are not well understood. To address this issue, we performed an unbiased comparative proteomic analysis of wild-type (WT) and Cav-1(-/-) null mammary stromal fibroblasts (MSFs). Our results show that plasminogen activator inhibitor type 1 and type 2 (PAI-1 and PAI-2) expression is significantly increased in Cav-1(-/-) MSFs. To establish a direct cause-effect relationship, we next generated immortalized human fibroblast lines stably overexpressing either PAI-1 or PAI-2. Importantly, PAI-1/2(+) fibroblasts promote the growth of MDA-MB-231 tumors (a human breast cancer cell line) in a murine xenograft model, without any increases in angiogenesis. Similarly, PAI-1/2(+) fibroblasts stimulate experimental metastasis of MDA-MB-231 cells using an in vivo lung colonization assay. Further mechanistic studies revealed that fibroblasts overexpressing PAI-1 or PAI-2 display increased autophagy (“self-eating”) and are sufficient to induce mitochondrial biogenesis/activity in adjacent cancer cells, in co-culture experiments. In xenografts, PAI-1/2(+) fibroblasts significantly reduce the apoptosis of MDA-MB-231 tumor cells. The current study provides further support for the “Autophagic Tumor Stroma Model of Cancer” and identifies a novel “extracellular matrix”-based signaling mechanism, by which a loss of stromal Cav-1 generates a metastatic phenotype. Thus, the secretion and remodeling of extracellular matrix components (such as PAI-1/2) can directly regulate both (1) autophagy in stromal fibroblasts and (2) epithelial tumor cell mitochondrial metabolism

Ketones and lactate “fuel” tumor growth and metastasis: Evidence that epithelial cancer cells use oxidative mitochondrial metabolism

Previously, we proposed a new model for understanding the “Warburg effect” in tumor metabolism. In this scheme, cancer-associated fibroblasts undergo aerobic glycolysis and the resulting energy-rich metabolites are then transferred to epithelial cancer cells, where they enter the TCA cycle, resulting in high ATP production via oxidative phosphorylation. We have termed this new paradigm “The Reverse Warburg Effect.” Here, we directly evaluate whether the end-products of aerobic glycolysis (3-hydroxy-butyrate and L-lactate) can stimulate tumor growth and metastasis, using MDA-MB-231 breast cancer xenografts as a model system. More specifically, we show that administration of 3-hydroxy-butyrate (a ketone body) increases tumor growth by ∼2.5-fold, without any measurable increases in tumor vascularization/angiogenesis. Both 3-hydroxy-butyrate and L-lactate functioned as chemo-attractants, stimulating the migration of epithelial cancer cells. Although L-lactate did not increase primary tumor growth, it stimulated the formation of lung metastases by ∼10-fold. Thus, we conclude that ketones and lactate fuel tumor growth and metastasis, providing functional evidence to support the “reverse Warburg effect.” Moreover, we discuss the possibility that it may be unwise to use lactate-containing i.v. solutions (such as lactated Ringer's or Hartmann's solution) in cancer patients, given the dramatic metastasis-promoting properties of L-lactate. Also, we provide evidence for the upregulation of oxidative mitochondrial metabolism and the TCA cycle in human breast cancer cells in vivo, via an informatics analysis of the existing raw transcriptional profiles of epithelial breast cancer cells and adjacent stromal cells. Lastly, our findings may explain why diabetic patients have an increased incidence of cancer, due to increased ketone production, and a tendency towards autophagy/mitophagy in their adipose tissue