A cell-type-specific role for murine Commd1 in liver inflammation

The transcription factor NF-kappaB plays a critical role in the inflammatory response and it has been implicated in various diseases, including non-alcoholic fatty liver disease (NAFLD). Although transient NF-kappaB activation may protect tissues from stress, a prolonged NF-kappaB activation can have a detrimental effect on tissue homeostasis and therefore accurate termination is crucial. Copper Metabolism MURR1 Domain-containing 1 (COMMD1), a protein with functions in multiple pathways, has been shown to suppress NF-kappaB activity. However, its action in controlling liver inflammation has not yet been investigated. To determine the cell-type-specific contribution of Commd1 to liver inflammation, we used hepatocyte and myeloid-specific Commd1-deficient mice. We also used a mouse model of NAFLD to study low-grade chronic liver inflammation: we fed the mice a high fat, high cholesterol (HFC) diet, which results in hepatic lipid accumulation accompanied by liver inflammation. Depletion of hepatocyte Commd1 resulted in elevated levels of the NF-kappaB transactivation subunit p65 (RelA) but, surprisingly, the level of liver inflammation was not aggravated. In contrast, deficiency of myeloid Commd1 exacerbated diet-induced liver inflammation. Unexpectedly we observed that hepatic and myeloid Commd1 deficiency in the mice both augmented hepatic lipid accumulation. The elevated levels of proinflammatory cytokines in myeloid Commd1-deficient mice might be responsible for the increased level of steatosis. This increase was not seen in hepatocyte Commd1-deficient mice, in which increased lipid accumulation appeared to be independent of inflammation. Our mouse models demonstrate a cell-type-specific role for Commd1 in suppressing liver inflammation and in the progression of NAFLD

Antibody validation and exploratory immunostaining for COMMD1 in human ovarian tumor samples.

<p><b>(A)</b> Representative immunohistochemical COMMD1 staining in paraffin embedded HEK293T and HeLa cells depleted for COMMD1. <b>(B)</b> HEK293T and HeLa cells were stably silenced for COMMD1 as shown by immunoblotting. (<b>C</b>) Observational immunostainings for COMMD1, including its control IgG₁ immunostaining in a consecutive slide, in HGSOC patient samples demonstrating either absent or presence of nuclear COMMD1. (<b>D</b>) Quantification workflow of immunohistochemical COMMD1 staining. Image analysis was performed using the ImageJ-based software package FIJI. DAB staining and hematoxylin staining were deconvoluted and images were subsequently converted into 8-bit gray scale images. Hematoxylin staining was used to define cytoplasm/nucleus boundaries. Vectors were subsequently used to measure DAB staining intensities across cells and quantify nuclear COMMD1 levels in relation to cytoplasmic levels. (B) Three ‘nuclear COMMD1-negative’ (n = 10 cells per tumor sample) and three ‘nuclear COMMD1-positive’ tumor samples were analyzed. Averages and standard deviations are indicated. Relative nuclear COMMD1 levels to cytoplasmic levels are plotted per tumor sample.</p

The COMMD family regulates plasma LDL levels and attenuates atherosclerosis through stabilizing the CCC complex in endosomal LDLR traffcking.

Rationale: COMMD (copper metabolism MURR1 domain)-containing proteins are a part of the CCC (COMMD-CCDC22 [coiled-coil domain containing 22]-CCDC93 [coiled-coil domain containing 93]) complex facilitating endosomal traffcking of cell surface receptors. Hepatic COMMD1 inactivation decreases CCDC22 and CCDC93 protein levels, impairs the recycling of the LDLR (low-density lipoprotein receptor), and increases plasma lowdensity lipoprotein cholesterol levels in mice. However, whether any of the other COMMD members function similarly as COMMD1 and whether perturbation in the CCC complex promotes atherogenesis remain unclear. Objective: The main aim of this study is to unravel the contribution of evolutionarily conserved COMMD proteins to plasma lipoprotein levels and atherogenesis. Methods and Results: Using liver-specifc Commd1, Commd6, or Commd9 knockout mice, we investigated the relation between the COMMD proteins in the regulation of plasma cholesterol levels. Combining biochemical and quantitative targeted proteomic approaches, we found that hepatic COMMD1, COMMD6, or COMMD9 defciency resulted in massive reduction in the protein levels of all 10 COMMDs. This decrease in COMMD protein levels coincided with destabilizing of the core (CCDC22, CCDC93, and chromosome 16 open reading frame 62 [C16orf62]) of the CCC complex, reduced cell surface levels of LDLR and LRP1 (LDLR-related protein 1), followed by increased plasma low-density lipoprotein cholesterol levels. To assess the direct contribution of the CCC core in the regulation of plasma cholesterol levels, Ccdc22 was deleted in mouse livers via CRISPR/Cas9-mediated somatic gene editing. CCDC22 defciency also destabilized the complete CCC complex and resulted in elevated plasma low-density lipoprotein cholesterol levels. Finally, we found that hepatic disruption of the CCC complex exacerbates dyslipidemia and atherosclerosis in ApoE3&lowast;Leiden mice. Conclusions: Collectively, these fndings demonstrate a strong interrelationship between COMMD proteins and the core of the CCC complex in endosomal LDLR traffcking. Hepatic disruption of either of these CCC components causes hypercholesterolemia and exacerbates atherosclerosis. Our results indicate that not only COMMD1 but all other COMMDs and CCC components may be potential targets for modulating plasma lipid levels in humans

A cell-type-specific role for murine Commd1 in liver inflammation

The transcription factor NF-κB plays a critical role in the inflammatory response and it has been implicated in various diseases, including non-alcoholic fatty liver disease (NAFLD). Although transient NF-κB activation may protect tissues from stress, a prolonged NF-κB activation can have a detrimental effect on tissue homeostasis and therefore accurate termination is crucial. Copper Metabolism MURR1 Domain-containing 1 (COMMD1), a protein with functions in multiple pathways, has been shown to suppress NF-κB activity. However, its action in controlling liver inflammation has not yet been investigated. To determine the cell-type-specific contribution of Commd1 to liver inflammation, we used hepatocyte and myeloid-specific Commd1-deficient mice. We also used a mouse model of NAFLD to study low-grade chronic liver inflammation: we fed the mice a high fat, high cholesterol (HFC) diet, which results in hepatic lipid accumulation accompanied by liver inflammation. Depletion of hepatocyte Commd1 resulted in elevated levels of the NF-κB transactivation subunit p65 (RelA) but, surprisingly, the level of liver inflammation was not aggravated. In contrast, deficiency of myeloid Commd1 exacerbated diet-induced liver inflammation. Unexpectedly we observed that hepatic and myeloid Commd1 deficiency in the mice both augmented hepatic lipid accumulation. The elevated levels of proinflammatory cytokines in myeloid Commd1-deficient mice might be responsible for the increased level of steatosis. This increase was not seen in hepatocyte Commd1-deficient mice, in which increased lipid accumulation appeared to be independent of inflammation. Our mouse models demonstrate a cell-type-specific role for Commd1 in suppressing liver inflammation and in the progression of NAFLD

Disease-specific and progression-free survival analysis in relation to nuclear COMMD1 expression in tumors of patients with advanced stage HGSOC from TMA2.

<p>Disease-specific and progression-free survival analysis in relation to nuclear COMMD1 expression in tumors of patients with advanced stage HGSOC from TMA2.</p

Increased nuclear COMMD1 expression in A2780 cells enhanced cisplatin sensitivity.

<p><b>(A)</b> Subcellular localization of COMMD1 in A2780 EV and A2780-COMMD1 cells determined by immunoblotting. Intensity of individual bands for COMMD1 was quantified using ImageLab software. After correction for tubulin or lamin A/C expression the relative COMMD1 expression in A2780-COMMD1 cells was determined. <b>(B)</b> A2780 EV and A2780 COMMD1 cells were stained for COMMD1 (green), and DNA (blue) and imaged by fluorescent microscopy. The scale bar represents 20 μm. <b>(C)</b> A2780 EV and A2780 COMMD1 cells were plated in 96-well plates and treated with indicated concentrations of cisplatin. After 72 hours of treatment, cells were incubated with MTT for 3 hours and the viability of cells was determined by colorimetric measurement. Data are shown from three independent experiments. Statistical significance was calculated using the Student's t-test. *: <i>P</i>< 0.05, **: <i>P</i> <0.01, ***: <i>P</i> <0.001. <b>(D)</b> Overexpression of COMMD1 in Peo14 cells augments cisplatin sensitivity. Control cells (Peo14 EV) and Peo14 cells stably overexpressing COMMD1-Flag (Peo14 COMMD1) were plated in 96-well plates and treated with indicated concentrations of cisplatin. After 72 hours of treatment, cells were incubated with MTT for 3 hours and the viability of cells was determined by colorimetric measurement. Data are shown from three independent experiments. Statistical significance was calculated using the Student's t-test. *: <i>P</i>< 0.05. (<b>E</b>) Subcellular localization of COMMD1 in Peo14-EV and Peo14-COMMD1 cells determined by immunoblotting. Intensity of individual bands for COMMD1 was quantified using ImageLab software. <b>(F)</b> Silencing of COMMD1 results in decreased sensitivity of A2780 cells to cisplatin. Control cells (EV) and COMMD1 silenced A2780 cells (KD) were plated in 96-well plates and treated with indicated concentrations of cisplatin. After 72 hours of treatment, cells were incubated with MTT for 3 hours and the viability of cells was determined by colorimetric measurement. Data are shown from three independent experiments. Statistical significance was calculated using the Student's t-test. *: <i>P</i>< 0.05</p

Nuclear COMMD1 Is Associated with Cisplatin Sensitivity in Ovarian Cancer

<div><p>Copper metabolism MURR1 domain 1 (COMMD1) protein is a multifunctional protein, and its expression has been correlated with patients’ survival in different types of cancer. <i>In vitro</i> studies revealed that COMMD1 plays a role in sensitizing cancer cell lines to cisplatin, however, the mechanism and its role in platinum sensitivity in cancer has yet to be established. We evaluated the role of COMMD1 in cisplatin sensitivity in A2780 ovarian cancer cells and the relation between COMMD1 expression and response to platinum-based therapy in advanced stage high-grade serous ovarian cancer (HGSOC) patients. We found that elevation of nuclear COMMD1 expression sensitized A2780 ovarian cancer cells to cisplatin-mediated cytotoxicity. This was accompanied by a more effective G₂/M checkpoint, and decreased protein expression of the DNA repair gene <i>BRCA1</i>, and the apoptosis inhibitor <i>BCL2</i>. Furthermore, COMMD1 expression was immunohistochemically analyzed in two tissue micro-arrays (TMAs), representing a historical cohort and a randomized clinical trial-based cohort of advanced stage HGSOC tumor specimens. Expression of COMMD1 was observed in all ovarian cancer samples, however, specifically nuclear expression of COMMD1 was only observed in a subset of ovarian cancers. In our historical cohort, nuclear COMMD1 expression was associated with an improved response to chemotherapy (OR = 0.167; <i>P</i> = 0.038), although this association could not be confirmed in the second cohort, likely due to sample size. Taken together, these results suggest that nuclear expression of COMMD1 sensitize ovarian cancer to cisplatin, possibly by modulating the G₂/M checkpoint and through controlling expression of genes involved in DNA repair and apoptosis.</p></div

Response to therapy for TMA1: logistic regression analysis for advanced stage HGSOC patients with ≥2 cm residual disease after primary surgery, and receiving platinum-based chemotherapy and a PFS of <6 months (n = 14) versus PFS of >18 months (n = 16).

<p>Response to therapy for TMA1: logistic regression analysis for advanced stage HGSOC patients with ≥2 cm residual disease after primary surgery, and receiving platinum-based chemotherapy and a PFS of <6 months (n = 14) versus PFS of >18 months (n = 16).</p

Model of how COMMD1 may influence responses to cisplatin.

<p>Model of how COMMD1 may influence responses to cisplatin.</p

Examples of COMMD1 levels and localization in ovarian cancer samples.

<p><b>(A)</b> Representative immunohistochemical stainings of cytoplasmic and nuclear COMMD1 are indicated. <b>(B)</b> Representative immunohistochemical stainings of cytoplasmic and nuclear COMMD1 of responders (n = 3) and non-responders (n = 3).</p