mARC1 in MASLD: Modulation of lipid accumulation in human hepatocytes and adipocytes.

BACKGROUND: Mutations in the gene MTARC1 (mitochondrial amidoxime-reducing component 1) protect carriers from metabolic dysfunction-associated steatohepatitis (MASH) and cirrhosis. MTARC1 encodes the mARC1 enzyme, which is localized to the mitochondria and has no known MASH-relevant molecular function. Our studies aimed to expand on the published human genetic mARC1 data and to observe the molecular effects of mARC1 modulation in preclinical MASH models. METHODS AND RESULTS: We identified a novel human structural variant deletion in MTARC1, which is associated with various biomarkers of liver health, including alanine aminotransferase levels. Phenome-wide Mendelian Randomization analyses additionally identified novel putatively causal associations between MTARC1 expression, and esophageal varices and cardiorespiratory traits. We observed that protective MTARC1 variants decreased protein accumulation in in vitro overexpression systems and used genetic tools to study mARC1 depletion in relevant human and mouse systems. Hepatocyte mARC1 knockdown in murine MASH models reduced body weight, liver steatosis, oxidative stress, cell death, and fibrogenesis markers. mARC1 siRNA treatment and overexpression modulated lipid accumulation and cell death consistently in primary human hepatocytes, hepatocyte cell lines, and primary human adipocytes. mARC1 depletion affected the accumulation of distinct lipid species and the expression of inflammatory and mitochondrial pathway genes/proteins in both in vitro and in vivo models. CONCLUSIONS: Depleting hepatocyte mARC1 improved metabolic dysfunction-associated steatotic liver disease-related outcomes. Given the functional role of mARC1 in human adipocyte lipid accumulation, systemic targeting of mARC1 should be considered when designing mARC1 therapies. Our data point to plasma lipid biomarkers predictive of mARC1 abundance, such as Ceramide 22:1. We propose future areas of study to describe the precise molecular function of mARC1, including lipid trafficking and subcellular location within or around the mitochondria and endoplasmic reticulum.

Systemically Administered Anti-uPAR Antibody Plasma and Lung ELF Pharmacokinetics Characterized by Minimal Lung PBPK Model.

Antibody-based therapeutics have recently gained keen attention for the treatment of pulmonary indications. However, systemically administered antibody exposure in the lungs needs to be better understood and remains a topic of interest. In this study, we evaluated the exposure of two different uPAR (urokinase-type plasminogen activator receptor) targeting full-length monoclonal IgGs in plasma and lung epithelial lining fluid (ELF) of mice after IP and IV administration. Antibody AK17 exhibited linear pharmacokinetics (PK) in plasma and ELF at 3 and 30 mg/kg single IV dose. The average plasma and ELF half-lives for AK17 and AK21 ranged between ~321-411 h and ~230-345 h, respectively, indicating sustained systemic and lung exposure of antibodies. The average ELF to the plasma concentration ratio of antibodies was ~0.01 and ~0.03 with IP and IV dosing, respectively, over 2 weeks post single dose. We simultaneously characterized plasma and ELF PK of antibody in mice by developing a minimal lung PBPK model for antibody. This model reasonably captured the plasma and ELF PK data while estimating three parameters. The model accounts for the convective transport of antibody into the tissues via blood and lymph flow. FcRn-mediated transcytosis was incorporated into the model for antibody distribution across the lung epithelial barrier. This model serves as a platform to predict the pulmonary PK of systemically administered antibodies and to support optimal dose selection for desired exposure in the lungs as the site of action.

PLCß2 Promotes VEGF-Induced Vascular Permeability.

BACKGROUND: Regulation of vascular permeability is critical to maintaining tissue metabolic homeostasis. VEGF (vascular endothelial growth factor) is a key stimulus of vascular permeability in acute and chronic diseases including ischemia reperfusion injury, sepsis, and cancer. Identification of novel regulators of vascular permeability would allow for the development of effective targeted therapeutics for patients with unmet medical need. METHODS: In vitro and in vivo models of VEGFA-induced vascular permeability, pathological permeability, quantitation of intracellular calcium release and cell entry, and phosphatidylinositol 4,5-bisphosphate levels were evaluated with and without modulation of PLC (phospholipase C) ß2. RESULTS: Global knock-out of PLCß2 in mice resulted in blockade of VEGFA-induced vascular permeability in vivo and transendothelial permeability in primary lung endothelial cells. Further work in an immortalized human microvascular cell line modulated with stable knockdown of PLCß2 recapitulated the observations in the mouse model and primary cell assays. Additionally, loss of PLCß2 limited both intracellular release and extracellular entry of calcium following VEGF stimulation as well as reduced basal and VEGFA-stimulated levels of phosphatidylinositol 4,5-bisphosphate compared to control cells. Finally, loss of PLCß2 in both a hyperoxia-induced lung permeability model and a cardiac ischemia:reperfusion model resulted in improved animal outcomes when compared with wild-type controls. CONCLUSIONS: The results implicate PLCß2 as a key positive regulator of VEGF-induced vascular permeability through regulation of both calcium flux and phosphatidylinositol 4,5-bisphosphate levels at the cellular level. Targeting of PLCß2 in a therapeutic setting may provide a novel approach to regulating vascular permeability in patients.

Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors.

BACKGROUND: Monoclonal antibodies have been used to effectively treat various tumors. We previously established a unique strategy to identify tumor specific antibodies by capturing B-cell response against breast tumor antigens from patient-derived sentinel lymph nodes. Initial application of this approach led to identification of a tumor specific single domain antibody. In this paper we optimized our previous strategy by generating heavy chain antibodies (HCAbs) to overcome the deficiencies of single domain antibodies. Here we identified and characterized a heavy chain antibody (HCAb2) that targets cell surface HSP90 antigen on breast tumor cells but not normal cells. METHODS: Eight HCAbs derived from 4 breast cancer patients were generated using an in vitro expression system. HCAbs were screened against normal breast cells (MCF10A, HMEC) and tumor cell lines (MCF7, MDA-MB-231) to identify cell surface targeting and tumor specific antibodies using flow cytometry and immunofluorescence. Results observed with cell lines were validated by screening a cohort of primary human breast normal and tumor tissues using immunofluorescence. Respective antigens for two HCAbs (HCAb1 and HCAb2) were identified using immunoprecipitation followed by mass spectrometry. Finally, we generated MDA-MB-231 xenograft tumors in NOD scid gamma mice and performed in vivo tumor targeting analysis of HCAb1 and HCAb2. RESULTS: Flow cytometry screen revealed that HCAb2 selectively bound to the surface of MDA-MB-231 cells in comparison to MCF10A and MCF7 cells. HCAb2 showed punctate membrane staining on MDA-MB-231 cells and preferential binding to human breast tumor tissues in comparison to normal breast tissues. In primary breast tumor tissues, HCAb2 showed positive binding to both E-cadherin positive and negative tumor cells. We identified and validated the target antigen of HCAb2 as Heat shock protein 90 (HSP90). HCAb2 also selectively targeted MDA-MB-231 xenograft tumor cells in vivo with little targeting to mouse normal tissues. Finally, HCAb2 specifically targeted calnexin negative xenograft tumor cells. CONCLUSIONS: From our screening methodology, we identified HCAb2 as a breast tumor specific heavy chain antibody targeting cell surface HSP90. HCAb2 also targeted MDA-MB-231 tumor cells in vivo suggesting that HCAb2 could be an ideal tumor targeting antibody.

Descriptive analysis of tumor cells with stem like phenotypes in metastatic and benign adrenal tumors.

BACKGROUND: Neuroblastoma (NB) comprises 7% of all childhood cancers. Here we report a descriptive analysis of key cellular markers that have "stem-like" properties which theoretically represents the self-renewing population of cells responsible for generating new tumor cells. Samples are obtained from freshly isolated tissue from nonmetastatic NB, metastatic NB, benign adrenal adenoma and a ganglioneuroma. In addition, in metastatic NB, descriptive analysis of the tumor cells after 3D culture as well as reanalysis of fresh tumor obtained after surgical excision posttreatment was performed. METHODS: Cells were isolated from primary tissue and characterized via immunohistochemistry and flow cytometry for markers associated with stem-like properties. In two patients, reanalysis was performed in freshly isolated tissue after chemotherapy. In three patients, freshly isolated tumors were cultured in 3 dimensions for 7-10 days and changes in stem-like marker expression were characterized. RESULTS: Flow analysis of metastatic NB revealed elevated levels of markers CD133, CD24, CD44, Oct4, CXCR4 and Nestin. In addition, some markers such as CD133 and CXCR4 maintained increased expression after chemotherapy. CONCLUSIONS: The expression profile of cells with "stem-like" properties has individual variability and differs depending on the tumor type. In metastatic NB, expression of "stem-like" markers Nestin, Oct4, and CXCR4 are maintained in a higher percentage of cells and this persists even after chemotherapy. In addition, culture of freshly isolated tissue maintained the individual expression profile of stem-like markers for at least 7 days.

AMP-Activated Protein Kinase α 2 Isoform Suppression in Primary Breast Cancer Alters AMPK Growth Control and Apoptotic Signaling.

Adenosine monophosphate-activated protein kinase (AMPK) is a metabolic regulator that promotes energy conservation and restoration when cells are exposed to nutrient stress. Given the high metabolic requirement of cancer cells, AMPK activation has been suggested as a potential preventative and therapeutic target. However, previous findings have shown that AMPK activity is diminished in some cancers. Expression of the 2 catalytic isoforms, AMPKα1 and AMPKα2, was evaluated in primary breast cancer and matched nontumor-adjacent tissue samples using immunohistochemistry. AMPK-dependent growth signaling events were examined in primary human mammary epithelial cells (HMECs) using RNAi to understand the importance of AMPKα2 in normal growth regulation. To test whether AMPKα2 would reinstate growth control and apoptotic mechanisms in breast cancer cells, metabolic stress assays and tumor xenografts were performed in MCF-7 cells, expressing low levels of AMPKα2, with stable transfection of either green fluorescent protein (GFP) or AMPKα2 expression constructs. AMPKα2 was found to be significantly suppressed in breast cancer tissue samples, whereas AMPKα1 was not. In normal HMECs, low glucose stress resulted in AMPK-driven growth inhibition. Interestingly, this response was ablated when AMPKα2 was silenced. Metabolic stress assays in MCF-7 cells indicated that AMPKα2 expression reduced both mTOR signaling and cyclin D1 expression, contributing to G1-phase cell cycle arrest. Cells expressing AMPKα2 underwent apoptosis more readily than GFP control cells. Xenograft studies demonstrated that MCF-7 tumors expressing AMPKα2 display reduced proliferation and increased apoptotic events. Furthermore, AMPKα2 xenografts exhibited diminished cyclin D1 levels along with an increased amount of nuclear p53, thereby implicating the AMPKα2-p53 signaling axis as a mediator of cell apoptosis. Together, these results highlight the significance of reduced AMPK activity contributing to human carcinogenesis and, specifically, the role of AMPKα2 with respect to its control of normal mammary epithelial cell growth and its reduced expression in breast cancer.

AMPKα2 Suppresses Murine Embryonic Fibroblast Transformation and Tumorigenesis.

AMP-activated kinase (AMPK) is a key metabolic sensor and stress signaling kinase. AMPK activity is known to suppress anabolic processes such as protein and lipid biosynthesis and promote energy-producing pathways including fatty acid oxidation, resulting in increased cellular energy. In addition, AMPK localizes to centrosomes during cell division, plays a role in cellular polarization, and directly targets p53, affecting apoptosis. Two distinct catalytic AMPKα isoforms exist: α1 and α2. Multiple reports indicate that both common and distinct functions exist for each of the 2 α isoforms. AMPK activation has been shown to repress tumor growth, and it has been suggested that AMPK may function as a metabolic tumor suppressor. To evaluate the potential role of each of the AMPKα isoforms in modulating cellular transformation, susceptibility to Ras-induced transformation was evaluated in normal murine embryonic fibroblasts (MEFs) obtained from genetically deleted AMPKα1- or AMPKα2-null mice. This study demonstrated that while AMPKα1 is the dominant AMPK isoform expressed in MEFs, only the AMPKα2-null MEFs displayed increased susceptibility to H-RasV12 transformation in vitro and tumorigenesis in vivo. Conversely, AMPKα1-null MEFs, which demonstrated compensation with increased expression of AMPKα2, displayed minimal transformation susceptibility, decreased cell survival, decreased cell proliferation, and increased apoptosis. Finally, this study demonstrates that AMPKα2 was selectively responsible for targeting p53, thus contributing to the suppression of transformation and tumorigenic mechanisms.

Revealing the role of phospholipase Cß3 in the regulation of VEGF-induced vascular permeability.

VEGF induces vascular permeability (VP) in ischemic diseases and cancer, leading to many pathophysiological consequences. The molecular mechanisms by which VEGF acts to induce hyperpermeability are poorly understood and in vivo models that easily facilitate real-time, genetic studies of VP do not exist. In the present study, we report a heat-inducible VEGF transgenic zebrafish (Danio rerio) model through which VP can be monitored in real time. Using this approach with morpholino-mediated gene knock-down and knockout mice, we describe a novel role of phospholipase Cß3 as a negative regulator of VEGF-mediated VP by regulating intracellular Ca2+ release. Our results suggest an important effect of PLCß3 on VP and provide a new model with which to identify genetic regulators of VP crucial to several disease processes.

Dietary energy availability affects primary and metastatic breast cancer and metformin efficacy.

Dietary energy restriction has been shown to repress both mammary tumorigenesis and aggressive mammary tumor growth in animal studies. Metformin, a caloric restriction mimetic, has a long history of safe use as an insulin sensitizer in diabetics and has been shown to reduce cancer incidence and cancer-related mortality in humans. To determine the potential impact of dietary energy availability and metformin therapy on aggressive breast tumor growth and metastasis, an orthotopic syngeneic model using triple negative 66cl4 tumor cells in Balb/c mice was employed. The effect of dietary restriction, a standard maintenance diet or a diet with high levels of free sugar, were tested for their effects on tumor growth and secondary metastases to the lung. Metformin therapy with the various diets indicated that metformin can be highly effective at suppressing systemic metabolic biomarkers such as IGF-1, insulin and glucose, especially in the high energy diet treated animals. Long-term metformin treatment demonstrated moderate yet significant effects on primary tumor growth, most significantly in conjunction with the high energy diet. When compared to the control diet, the high energy diet promoted tumor growth, expression of the inflammatory adipokines leptin and resistin, induced lung priming by bone marrow-derived myeloid cells and promoted metastatic potential. Metformin had no effect on adipokine expression or the development of lung metastases with the standard or the high energy diet. These data indicate that metformin may have tumor suppressing activity where a metabolic phenotype of high fuel intake, metabolic syndrome, and diabetes exist, but may have little or no effect on events controlling the metastatic niche driven by proinflammatory events.

Identification of novel tumor antigens with patient-derived immune-selected antibodies.

The identification of tumor antigens capable of eliciting an immune response in vivo may be an effective method to identify therapeutic cancer targets. We have developed a method to identify such antigens using frozen tumor-draining lymph node samples from breast cancer patients. Immune responses in tumor-draining lymph nodes were identified by immunostaining lymph node sections for B-cell markers (CD20&CD23) and Ki67 which revealed cell proliferation in germinal center zones. Antigen-dependent somatic hypermutation (SH) and clonal expansion (CE) were present in heavy chain variable (VH) domain cDNA clones obtained from these germinal centers, but not from Ki67 negative germinal centers. Recombinant VH single-domain antibodies were used to screen tumor proteins and affinity select potential tumor antigens. Neuroplastin (NPTN) was identified as a candidate breast tumor antigen using proteomic identification of affinity selected tumor proteins with a recombinant VH single chain antibody. NPTN was found to be highly expressed in approximately 20% of invasive breast carcinomas and 50% of breast carcinomas with distal metastasis using a breast cancer tissue array. Additionally, NPTN over-expression in a breast cancer cell line resulted in a significant increase in tumor growth and angiogenesis in vivo which was related to increased VEGF production in the transfected cells. These results validate NPTN as a tumor-associated antigen which could promote breast tumor growth and metastasis if aberrantly expressed. These studies also demonstrate that humoral immune responses in tumor-draining lymph nodes can provide antibody reagents useful in identifying tumor antigens with applications for biomarker screening, diagnostics and therapeutic interventions.

Therapeutic metformin/AMPK activation promotes the angiogenic phenotype in the ERalpha negative MDA-MB-435 breast cancer model.

Metformin, a first line treatment for type 2 diabetes, has been implicated as a potential anti-neoplastic agent for breast cancers as well as other cancers. Metformin is known to work in part through the activation of AMP-dependent kinase (AMPK). AMPK is a key regulator of cellular energy homeostasis, especially under stress conditions where biosynthetic pathways are blocked by the phosphorylation of downstream AMPK substrates. Stimulation of AMPK by metformin resulted in a significant repression of cell proliferation and active MAPK1/2 in both estrogen receptor alpha (ERalpha) negative (MDA-MB-231, MDA-MB-435) and positive (MCF-7, T47D) human breast cancer cell lines. However, when ERalpha negative MDA-MB-435 cells were treated with metformin, they demonstrated increased expression of vascular endothelial growth factor (VEGF) in an AMPK dependent manner; while the ERalpha positive MCF-7 cells did not. Systemic therapy with metformin was tested for efficacy in an orthotopic model of ERalpha negative breast cancer performed in athymic nude mice. Surprisingly, metformin therapy significantly improved tumorigenic progression as compared to untreated controls. The metformin-treated group showed increased VEGF expression, intratumoral microvascular density and reduced necrosis. Metformin treatment was sufficient, however, to reduce systemic IGF-1 and the proliferation rate of tumor cells in vascularized regions. The data presented here suggests that, although metformin significantly represses breast cancer cell growth in vitro, the efficacy with respect to its therapeutic application for ERalpha negative breast cancer lesions in vivo may result in promotion of the angiogenic phenotype and increased tumorigenic progression.

Regulation of ovarian cancer cell viability and sensitivity to cisplatin by progesterone receptor membrane component-1.

CONTEXT: Progesterone (P4) influences ovarian cancer cells by an unknown mechanism. OBJECTIVE: The objective was to determine whether P4 acts through progesterone receptor membrane component-1 (PGRMC1) in ovarian cancers. DESIGN, SETTING AND PATIENTS: Archival tissue and cDNA provided by OriGene were used for expression studies. In vitro experiments were conducted with Ovcar-3 cells. MAIN OUTCOME MEASURES: PCR, Western blot, and immunohistochemistry were used to measure expression of PGRMC1 and nuclear progesterone receptor (PGR). PGRMC1's role in regulating the viability of ovarian cancers was assessed by overexpressing PGRMC1, depleting PGRMC1 using small interfering RNA, and attenuating PGRMC1's action with a blocking antibody. Apoptosis was determined by 4',6'-diamino-2-phenylindole staining. RESULTS: PGRMC1 mRNA increased and PGR mRNA decreased in advanced stages of ovarian cancer. Unlike PGR, PGRMC1 was expressed in virtually every cancer cell within the tumor. A similar relationship between PGRMC1 and PGR was observed in Ovcar-3 cells. In these cells P4 suppressed apoptosis induced by either serum withdrawal or cisplatin (CDDP). Moreover, in the presence of P4, the following occurs: 1) overexpression of PGRMC1 reduces the effectiveness of CDDP, 2) depletion of PGRMC1 with small interfering RNA enhances the effects of CDDP, and 3) PGRMC1 antibody treatment increases the apoptotic response to CDDP. CONCLUSIONS: These findings indicate that PGRMC1 plays an important role in promoting ovarian cancer cell viability and that attenuating PGRMC1's action makes the ovarian cancer cells more sensitive to CDDP. These data suggest that targeted depletion of PGRMC1 could be useful as an adjunct to CDDP therapy.

Double-stranded RNA-binding protein regulates vascular endothelial growth factor mRNA stability, translation, and breast cancer angiogenesis.

Vascular endothelial growth factor (VEGF) is a key angiogenic factor expressed under restricted nutrient and oxygen conditions in most solid tumors. The expression of VEGF under hypoxic conditions requires transcription through activated hypoxia-inducible factor 1 (HIF-1), increased mRNA stability, and facilitated translation. This study identified double-stranded RNA-binding protein 76/NF90 (DRBP76/NF90), a specific isoform of the DRBP family, as a VEGF mRNA-binding protein which plays a key role in VEGF mRNA stability and protein synthesis under hypoxia. The DRBP76/NF90 protein binds to a human VEGF 3' untranslated mRNA stability element. RNA interference targeting the DRBP76/NF90 isoform limited hypoxia-inducible VEGF mRNA and protein expression with no change in HIF-1-dependent transcriptional activity. Stable repression of DRBP76/NF90 in MDA-MB-435 breast cancer cells demonstrated reduced polysome-associated VEGF mRNA levels under hypoxic conditions and reduced mRNA stability. Transient overexpression of the DRBP76/NF90 protein increased both VEGF mRNA and protein levels synthesized under normoxic and hypoxic conditions. Cells with stable repression of the DRBP76/NF90 isoform showed reduced tumorigenic and angiogenic potential in an orthotopic breast tumor model. These data demonstrate that the DRBP76/NF90 isoform facilitates VEGF expression by promoting VEGF mRNA loading onto polysomes and translation under hypoxic conditions, thus promoting breast cancer growth and angiogenesis in vivo.