The phospholipid transporter PITPNC1 links KRAS to MYC to prevent autophagy in lung and pancreatic cancer.

BACKGROUND: The discovery of functionally relevant KRAS effectors in lung and pancreatic ductal adenocarcinoma (LUAD and PDAC) may yield novel molecular targets or mechanisms amenable to inhibition strategies. Phospholipids availability has been appreciated as a mechanism to modulate KRAS oncogenic potential. Thus, phospholipid transporters may play a functional role in KRAS-driven oncogenesis. Here, we identified and systematically studied the phospholipid transporter PITPNC1 and its controlled network in LUAD and PDAC. METHODS: Genetic modulation of KRAS expression as well as pharmacological inhibition of canonical effectors was completed. PITPNC1 genetic depletion was performed in in vitro and in vivo LUAD and PDAC models. PITPNC1-deficient cells were RNA sequenced, and Gene Ontology and enrichment analyses were applied to the output data. Protein-based biochemical and subcellular localization assays were run to investigate PITPNC1-regulated pathways. A drug repurposing approach was used to predict surrogate PITPNC1 inhibitors that were tested in combination with KRASG12C inhibitors in 2D, 3D, and in vivo models. RESULTS: PITPNC1 was increased in human LUAD and PDAC, and associated with poor patients' survival. PITPNC1 was regulated by KRAS through MEK1/2 and JNK1/2. Functional experiments showed PITPNC1 requirement for cell proliferation, cell cycle progression and tumour growth. Furthermore, PITPNC1 overexpression enhanced lung colonization and liver metastasis. PITPNC1 regulated a transcriptional signature which highly overlapped with that of KRAS, and controlled mTOR localization via enhanced MYC protein stability to prevent autophagy. JAK2 inhibitors were predicted as putative PITPNC1 inhibitors with antiproliferative effect and their combination with KRASG12C inhibitors elicited a substantial anti-tumour effect in LUAD and PDAC. CONCLUSIONS: Our data highlight the functional and clinical relevance of PITPNC1 in LUAD and PDAC. Moreover, PITPNC1 constitutes a new mechanism linking KRAS to MYC, and controls a druggable transcriptional network for combinatorial treatments.

Carbon Dot Therapeutic Platforms: Administration, Distribution, Metabolism, Excretion, Toxicity, and Therapeutic Potential.

Ultrasmall nanoparticles are often grouped under the broad umbrella term of "nanoparticles" when reported in the literature. However, for biomedical applications, their small sizes give them intimate interactions with biological species and endow them with unique functional physiochemical properties. Carbon quantum dots (CQDs) are an emerging class of ultrasmall nanoparticles which have demonstrated considerable biocompatibility and have been employed as potent theragnostic platforms. These particles find application for increasing drug solubility and targeting, along with facilitating the passage of drugs across impermeable membranes (i.e., blood brain barrier). Further functionality can be triggered by various environmental conditions or external stimuli (i.e., pH, temperature, near Infrared (NIR) light, ultrasound), and their intrinsic fluorescence is valuable for diagnostic applications. The focus of this review is to shed light on the therapeutic potential of CQDs and identify how they travel through the body, reach their site of action, administer therapeutic effect, and are excreted. Investigation into their toxicity and compatibility with larger nanoparticle carriers is also examined. The future of CQDs for theragnostic applications is promising due to their multifunctional attributes and documented biocompatibility. As nanomaterial platforms become more commonplace in clinical treatments, the commercialization of CQD therapeutics is anticipated.

N6-methyladenosine marks primary microRNAs for processing.

The first step in the biogenesis of microRNAs is the processing of primary microRNAs (pri-miRNAs) by the microprocessor complex, composed of the RNA-binding protein DGCR8 and the type III RNase DROSHA. This initial event requires recognition of the junction between the stem and the flanking single-stranded RNA of the pri-miRNA hairpin by DGCR8 followed by recruitment of DROSHA, which cleaves the RNA duplex to yield the pre-miRNA product. While the mechanisms underlying pri-miRNA processing have been determined, the mechanism by which DGCR8 recognizes and binds pri-miRNAs, as opposed to other secondary structures present in transcripts, is not understood. Here we find in mammalian cells that methyltransferase-like 3 (METTL3) methylates pri-miRNAs, marking them for recognition and processing by DGCR8. Consistent with this, METTL3 depletion reduced the binding of DGCR8 to pri-miRNAs and resulted in the global reduction of mature miRNAs and concomitant accumulation of unprocessed pri-miRNAs. In vitro processing reactions confirmed the sufficiency of the N(6)-methyladenosine (m(6)A) mark in promoting pri-miRNA processing. Finally, gain-of-function experiments revealed that METTL3 is sufficient to enhance miRNA maturation in a global and non-cell-type-specific manner. Our findings reveal that the m(6)A mark acts as a key post-transcriptional modification that promotes the initiation of miRNA biogenesis.

PTPRN2 and PLCß1 promote metastatic breast cancer cell migration through PI(4,5)P2-dependent actin remodeling.

Altered abundance of phosphatidyl inositides (PIs) is a feature of cancer. Various PIs mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN2 and PLCß1 enzymatically reduce plasma membrane PI(4,5)P2 levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI(4,5)P2 abundance by these enzymes releases the PI(4,5)P2-binding protein cofilin from its inactive membrane-associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid-dependent sequestration of an actin-remodeling factor.

A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells.

Metastatic progression of cancer is a complex and clinically daunting process. We previously identified a set of human microRNAs (miRNAs) that robustly suppress breast cancer metastasis to lung and bone and which display expression levels that predict human metastasis. Although these findings revealed miRNAs as suppressors of cell-autonomous metastatic phenotypes, the roles of non-coding RNAs in non-cell-autonomous cancer progression processes remain unknown. Here we reveal that endogenous miR-126, an miRNA silenced in a variety of common human cancers, non-cell-autonomously regulates endothelial cell recruitment to metastatic breast cancer cells, in vitro and in vivo. It suppresses metastatic endothelial recruitment, metastatic angiogenesis and metastatic colonization through coordinate targeting of IGFBP2, PITPNC1 and MERTK--novel pro-angiogenic genes and biomarkers of human metastasis. Insulin-like growth factor binding protein 2 (IGFBP2) secreted by metastatic cells recruits endothelia by modulating IGF1-mediated activation of the IGF type-I receptor on endothelial cells; whereas c-Mer tyrosine kinase (MERTK) receptor cleaved from metastatic cells promotes endothelial recruitment by competitively antagonizing the binding of its ligand GAS6 to endothelial MERTK receptors. Co-injection of endothelial cells with breast cancer cells non-cell-autonomously rescues their miR-126-induced metastatic defect, revealing a novel and important role for endothelial interactions in metastatic initiation. Through loss-of-function and epistasis experiments, we delineate an miRNA regulatory network's individual components as novel and cell-extrinsic regulators of endothelial recruitment, angiogenesis and metastatic colonization. We also identify the IGFBP2/IGF1/IGF1R and GAS6/MERTK signalling pathways as regulators of cancer-mediated endothelial recruitment. Our work further reveals endothelial recruitment and endothelial interactions in the tumour microenvironment to be critical features of metastatic breast cancer.

Differential transendothelial transport of adiponectin complexes.

BACKGROUND: Adiponectin's effects on systemic physiology and cell-specific responses are well-defined, but little is known about how this insulin-sensitizing and anti-inflammatory adipokine reaches its target cells. All molecules face active and passive transport limitations, but adiponectin is particularly noteworthy due to the diverse size range and high molecular weights of its oligomers. Additionally, its metabolic target organs possess a range of endothelial permeability. METHODS: Full-length recombinant murine adiponectin was produced and oligomer fractions isolated by gel filtration. Adiponectin complex sizes were measured by dynamic light scattering to determine Stokes radii. Transendothelial transport of purified oligomers was quantitatively assessed under a number of different conditions in vitro using murine endothelial cells and in vivo using several mouse models of altered endothelial function. RESULTS: Adiponectin oligomers exhibit large transport radii that limit transendothelial transport. Oligomerization is a significant determinant of flux across endothelial monolayers in vitro; low molecular weight adiponectin is preferentially transported. In vivo sampled sera from the heart, liver, and tail vein demonstrated significantly different complex distribution of lower molecular weight oligomers. Pharmacological interventions, such as PPARγ agonist treatment, differentially affect adiponectin plasma clearance and tissue uptake. Exercise induces enhanced adiponectin uptake to oxidative skeletal muscles, wherein adiponectin potently lowers ceramide levels. In total, endothelial barriers control adiponectin transport in a cell- and tissue-specific manner. CONCLUSIONS: Adiponectin oligomer efficacy in a given tissue may therefore be endothelial transport mediated. Targeting endothelial dysfunction in the metabolic syndrome through exercise and pharmaceuticals may afford an effective approach to increasing adiponectin's beneficial effects.

Chronic intermittent hypoxia induces atherosclerosis via activation of adipose angiopoietin-like 4.

RATIONALE: Obstructive sleep apnea is a risk factor for dyslipidemia and atherosclerosis, which have been attributed to chronic intermittent hypoxia (CIH). Intermittent hypoxia inhibits a key enzyme of lipoprotein clearance, lipoprotein lipase, and up-regulates a lipoprotein lipase inhibitor, angiopoietin-like 4 (Angptl4), in adipose tissue. The effects and mechanisms of Angptl4 up-regulation in sleep apnea are unknown. OBJECTIVES: To examine whether CIH induces dyslipidemia and atherosclerosis by increasing adipose Angptl4 via hypoxia-inducible factor-1 (HIF-1). METHODS: ApoE(-/-) mice were exposed to intermittent hypoxia or air for 4 weeks while being treated with Angptl4-neutralizing antibody or vehicle. MEASUREMENTS AND MAIN RESULTS: In vehicle-treated mice, hypoxia increased adipose Angptl4 levels, inhibited adipose lipoprotein lipase, increased fasting levels of plasma triglycerides and very low density lipoprotein cholesterol, and increased the size of atherosclerotic plaques. The effects of CIH were abolished by the antibody. Hypoxia-induced increases in plasma fasting triglycerides and adipose Angptl4 were not observed in mice with germline heterozygosity for a HIF-1α knockout allele. Transgenic overexpression of HIF-1α in adipose tissue led to dyslipidemia and increased levels of adipose Angptl4. In cultured adipocytes, constitutive expression of HIF-1α increased Angptl4 levels, which was abolished by siRNA. Finally, in obese patients undergoing bariatric surgery, the severity of nocturnal hypoxemia predicted Angptl4 levels in subcutaneous adipose tissue. CONCLUSIONS: HIF-1-mediated increase in adipose Angptl4 and the ensuing lipoprotein lipase inactivation may contribute to atherosclerosis in patients with sleep apnea.

PAQR4 regulates adipocyte function and systemic metabolic health by mediating ceramide levels.

PAQR4 is an orphan receptor in the PAQR family with an unknown function in metabolism. Here, we identify a critical role of PAQR4 in maintaining adipose tissue function and whole-body metabolic health. We demonstrate that expression of Paqr4 specifically in adipocytes, in an inducible and reversible fashion, leads to partial lipodystrophy, hyperglycaemia and hyperinsulinaemia, which is ameliorated by wild-type adipose tissue transplants or leptin treatment. By contrast, deletion of Paqr4 in adipocytes improves healthy adipose remodelling and glucose homoeostasis in diet-induced obesity. Mechanistically, PAQR4 regulates ceramide levels by mediating the stability of ceramide synthases (CERS2 and CERS5) and, thus, their activities. Overactivation of the PQAR4-CERS axis causes ceramide accumulation and impairs adipose tissue function through suppressing adipogenesis and triggering adipocyte de-differentiation. Blocking de novo ceramide biosynthesis rescues PAQR4-induced metabolic defects. Collectively, our findings suggest a critical function of PAQR4 in regulating cellular ceramide homoeostasis and targeting PAQR4 offers an approach for the treatment of metabolic disorders.

Limiting mitochondrial plasticity by targeting DRP1 induces metabolic reprogramming and reduces breast cancer brain metastases.

Disseminated tumor cells with metabolic flexibility to utilize available nutrients in distal organs persist, but the precise mechanisms that facilitate metabolic adaptations remain unclear. Here we show fragmented mitochondrial puncta in latent brain metastatic (Lat) cells enable fatty acid oxidation (FAO) to sustain cellular bioenergetics and maintain redox homeostasis. Depleting the enriched dynamin-related protein 1 (DRP1) and limiting mitochondrial plasticity in Lat cells results in increased lipid droplet accumulation, impaired FAO and attenuated metastasis. Likewise, pharmacological inhibition of DRP1 using a small-molecule brain-permeable inhibitor attenuated metastatic burden in preclinical models. In agreement with these findings, increased phospho-DRP1 expression was observed in metachronous brain metastasis compared with patient-matched primary tumors. Overall, our findings reveal the pivotal role of mitochondrial plasticity in supporting the survival of Lat cells and highlight the therapeutic potential of targeting cellular plasticity programs in combination with tumor-specific alterations to prevent metastatic recurrences.

C/EBPB-dependent adaptation to palmitic acid promotes tumor formation in hormone receptor negative breast cancer.

Epidemiological studies have established a positive association between obesity and the incidence of postmenopausal breast cancer. Moreover, it is known that obesity promotes stem cell-like properties of breast cancer cells. However, the cancer cell-autonomous mechanisms underlying this correlation are not well defined. Here we demonstrate that obesity-associated tumor formation is driven by cellular adaptation rather than expansion of pre-existing clones within the cancer cell population. While there is no correlation with specific mutations, cellular adaptation to obesity is governed by palmitic acid (PA) and leads to enhanced tumor formation capacity of breast cancer cells. This process is governed epigenetically through increased chromatin occupancy of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPB). Obesity-induced epigenetic activation of C/EBPB regulates cancer stem-like properties by modulating the expression of key downstream regulators including CLDN1 and LCN2. Collectively, our findings demonstrate that obesity drives cellular adaptation to PA drives tumor initiation in the obese setting through activation of a C/EBPB dependent transcriptional network.

Cellular mechanisms linking cancers to obesity.

Obesity is epidemiologically linked to 13 forms of cancer. The local and systemic obese environment is complex and likely affect tumors through multiple avenues. This includes modulation of cancer cell phenotypes and the composition of the tumor microenvironment. A molecular understanding of how obesity links to cancer holds promise for identifying candidate genes for targeted therapy for obese cancer patient. Herein, we review both the cell-autonomous and non-cell-autonomous mechanisms linking obesity and cancer as well as provide an overview of the mouse model systems applied to study this.

High-dimensional immunotyping of tumors grown in obese and non-obese mice.

Obesity is a disease characterized by chronic low-grade systemic inflammation and has been causally linked to the development of 13 cancer types. Several studies have been undertaken to determine whether tumors evolving in obese environments adapt differential interactions with immune cells and whether this can be connected to disease outcome. Most of these studies have been limited to single-cell lines and tumor models and analysis of limited immune cell populations. Given the multicellular complexity of the immune system and its dysregulation in obesity, we applied high-dimensional suspension mass cytometry to investigate how obesity affects tumor immunity. We used a 36-marker immune-focused mass cytometry panel to interrogate the immune landscape of orthotopic syngeneic mouse models of pancreatic and breast cancer. Unanchored batch correction was implemented to enable simultaneous analysis of tumor cohorts to uncover the immunotypes of each cancer model and reveal remarkably model-specific immune regulation. In the E0771 breast cancer model, we demonstrate an important link to obesity with an increase in two T-cell-suppressive cell types and a decrease in CD8 T cells.

Golgi-Localized PAQR4 Mediates Antiapoptotic Ceramidase Activity in Breast Cancer.

The metabolic network of sphingolipids plays important roles in cancer biology. Prominent sphingolipids include ceramides and sphingosine-1-phosphate that regulate multiple aspects of growth, apoptosis, and cellular signaling. Although a significant number of enzymatic regulators of the sphingolipid pathway have been described in detail, many remained poorly characterized. Here we applied a patient-derived systemic approach to identify and molecularly define progestin and adipoQ receptor family member IV (PAQR4) as a Golgi-localized ceramidase. PAQR4 was approximately 5-fold upregulated in breast cancer compared with matched control tissue and its overexpression correlated with disease-specific survival rates in breast cancer. Induction of PAQR4 in breast tumors was found to be subtype-independent and correlated with increased ceramidase activity. These findings establish PAQR4 as Golgi-localized ceramidase required for cellular growth in breast cancer. SIGNIFICANCE: Induction of and cellular dependency on de novo sphingolipid synthesis via PAQR4 highlights a central vulnerability in breast cancer that may serve as a viable therapeutic target.

AXL Is a Driver of Stemness in Normal Mammary Gland and Breast Cancer.

The receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony formation and self-maintenance activities while promoting terminal differentiation in vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl + cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when co-opted, maintains acquired stemness in breast cancer cells.

Human endotrophin as a driver of malignant tumor growth.

We have previously reported that the carboxy-terminal proteolytic cleavage product of the COL6α3 chain that we refer to as "endotrophin" has potent effects on transformed mammary ductal epithelial cells in rodents. Endotrophin (ETP) is abundantly expressed in adipose tissue. It is a chemoattractant for macrophages, exerts effects on endothelial cells and through epithelial-mesenchymal transition (EMT) enhances progression of tumor cells. In a recombinant form, human endotrophin exerts similar effects on human macrophages and endothelial cells as its rodent counterpart. It enhances EMT in human breast cancer cells and upon overexpression in tumor cells, the cells become chemoresistant. Here, we report the identification of endotrophin from human plasma. It is circulating at higher levels in breast cancer patients. We have developed neutralizing monoclonal antibodies against human endotrophin and provide evidence for the effectiveness of these antibodies to curb tumor growth and enhance chemosensitivity in a nude mouse model carrying human tumor cell lesions. Combined, the data validate endotrophin as a viable target for anti-tumor therapy for human breast cancer and opens the possibility for further use of these new reagents for anti-fibrotic approaches in liver, kidney, bone marrow and adipose tissue.

Upregulated PDK4 expression is a sensitive marker of increased fatty acid oxidation.

Fatty acid oxidation is a central fueling pathway for mitochondrial ATP production. Regulation occurs through multiple nutrient- and energy-sensitive molecular mechanisms. We explored if upregulated mRNA expression of the mitochondrial enzyme pyruvate dehydrogenase kinase 4 (PDK4) may be used as a surrogate marker of increased mitochondrial fatty acid oxidation, by indicating an overall shift from glucose to fatty acids as the preferred oxidation fuel. The association between fatty acid oxidation and PDK4 expression was studied in different contexts of metabolic adaption. In rats treated with the modified fatty acid tetradecylthioacetic acid (TTA), Pdk4 was upregulated simultaneously with fatty acid oxidation genes in liver and heart, whereas muscle and white adipose tissue remained unaffected. In MDA-MB-231 cells, fatty acid oxidation increased nearly three-fold upon peroxisome proliferator-activated receptor α (PPARα, PPARA) overexpression, and four-fold upon TTA-treatment. PDK4 expression was highly increased under these conditions. Further, overexpression of PDK4 caused increased fatty acid oxidation in these cells. Pharmacological activators of PPARα and AMPK had minor effects, while the mTOR inhibitor rapamycin potentiated the effect of TTA. There were minor changes in mitochondrial respiration, glycolytic function, and mitochondrial biogenesis under conditions of increased fatty acid oxidation. TTA was found to act as a mild uncoupler, which is likely to contribute to the metabolic effects. Repeated experiments with HeLa cells supported these findings. In summary, PDK4 upregulation implies an overarching metabolic shift towards increased utilization of fatty acids as energy fuel, and thus constitutes a sensitive marker of enhanced fatty acid oxidation.

Plasma adiponectin complexes have distinct biochemical characteristics.

Adipocytes release the secretory protein adiponectin in a number of different higher-order complexes. Once synthesized and assembled in the secretory pathway of the adipocyte, these complexes circulate as biochemically distinct and stable entities with little evidence of interchange between the different forms that include a high-molecular-weight (HMW) species, a hexamer (low-molecular-weight form), and a trimeric form of the complexes. Here, we validate a high-resolution gel filtration method that reproducibly separates the three complexes in recombinant adiponectin and adiponectin from human and murine samples. We demonstrate that the HMW form is prominently reduced in male vs. female subjects and in obese, insulin-resistant vs. lean, insulin-sensitive individuals. A direct comparison of human and mouse adiponectin demonstrates that the trimer is generally more abundant in human serum. Furthermore, when the production of adiponectin is reduced, either by obesity or in mice carrying only a single functional allele of the adiponectin locus, then the amount of the HMW form is selectively reduced in circulation. The complex distribution of adiponectin can be regulated in several ways. Both mouse and human HMW adiponectin are very stable under basic conditions but are exquisitely labile under acidic conditions below pH 7. Murine and human adiponectin HMW forms also display differential susceptibility to the presence of calcium in the buffer. A mutant form of adiponectin unable to bind calcium is less susceptible to changes in calcium concentrations. However, the lack of calcium binding results in a destabilization of the structure. Disulfide bond formation (at position C39) is also important for complex formation. A mutant form of adiponectin lacking C39 prominently forms HMW and trimer but not the low-molecular-weight form. Injection of adiponectin with a fluorescent label reveals that over time, the various complexes do not interconvert in vivo. The stability of adiponectin complexes highlights that the production and secretion of these forms from fat cells has a major influence on the circulating levels of each complex.

The adipocyte as an endocrine cell.

Adipose tissue contains many cell types. Among the more abundant are adipocytes, preadipocytes, immune cells, and endothelial cells. During times of excess caloric intake, these cells have to adjust and remodel to accommodate the increased demand for triglyceride storage. Based on a comprehensive analysis of the total adipose tissue secretome, this article focuses on three areas of adipokine biology: (1) How does the adipocyte interact with the extracellular matrix over the course of obesity? (2) Does the adipocyte, per se, play a role in the innate immune response? (3) How is the angiogenic profile of adipose tissue linked to the development of insulin resistance? The authors present a comprehensive overview of all of the currently available secreted adipose tissue products that have been identified at the protein level.