miR-126 Decreases Proliferation and Mammosphere Formation of MCF-7 and Predicts Prognosis of ER+ Breast Cancer.

Breast cancer (BC) is a major health burden that affects over one million women each year. It is the most prevalent cancer in women and the number one cancer killer of them worldwide. Of all BC subtypes, estrogen receptor-positive (ER+) BC is the most commonly diagnosed. The objective of this study is to investigate the contribution of miR-126 in the tumorigenesis of ER+ BC. miR-126 was downregulated in ER+ BC tissues from young breast cancer patients, as shown through miRNA microarray analysis and RT-qPCR. Subsequently, the effect of the modulation of miR-126 levels on the proliferation, cell cycle progression, and spheres formation of the ER+ BC cell line, MCF-7, was assessed by MTT assay, PI analysis, and mammosphere formation assay, respectively. miR-126 overexpression significantly decreased MCF-7 proliferation and mammosphere-forming ability, but did not affect cell cycle progression. Then, in silico analysis determined SLC7A5, PLXNB2, CRK, PLK2, SPRED1, and IRS1 as potential targets of miR-126. RT-qPCR data showed that miR-126 overexpression significantly downregulated SLC7A5 and PLXNB2 mRNA levels in MCF-7. Finally, in silico survival analysis showed that high expression of miR-126 or low expression of SLC7A5 correlated with better overall survival (OS) of ER+ BC patients. Overall, our study suggests that miR-126 might play a tumor suppressor role in ER+ BC. miR-126 and SLC7A5 might also be considered potential prognostic biomarkers in ER+ BC.

Metabolic dysregulation and emerging therapeutical targets for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive human cancer with increasing incidence worldwide. Multiple efforts have been made to explore pharmaceutical therapies to treat HCC, such as targeted tyrosine kinase inhibitors, immune based therapies and combination of chemotherapy. However, limitations exist in current strategies including chemoresistance for instance. Tumor initiation and progression is driven by reprogramming of metabolism, in particular during HCC development. Recently, metabolic associated fatty liver disease (MAFLD), a reappraisal of new nomenclature for non-alcoholic fatty liver disease (NAFLD), indicates growing appreciation of metabolism in the pathogenesis of liver disease, including HCC, thereby suggesting new strategies by targeting abnormal metabolism for HCC treatment. In this review, we introduce directions by highlighting the metabolic targets in glucose, fatty acid, amino acid and glutamine metabolism, which are suitable for HCC pharmaceutical intervention. We also summarize and discuss current pharmaceutical agents and studies targeting deregulated metabolism during HCC treatment. Furthermore, opportunities and challenges in the discovery and development of HCC therapy targeting metabolism are discussed.

Neuronal allodynic mechanisms of Slc7a5 (LAT1) in the spared nerve injury rodent model of neuropathic pain.

High-impact chronic pain is suffered by 1 in 5 patients in the USA and globally. Effective, non-addictive, non-opioid therapeutics are urgently needed for the treatment of chronic pain. Slc7a5 (Lat1), also known as system L-neutral amino acid transporter, is involved in a number of physiological processes related to inflammation. Transcriptomics studies have shown that Slc7a5 and its binding partner Slc3a2 are expressed in neurons of the dorsal root ganglia (DRG) and spinal dorsal horn, which are critical to the initiation and maintenance of nociception and pathophysiology of chronic pain. In addition, Slc7a5 is a transporter for the first-line anti-allodynic gabapentinoid drugs and binds to ion channels implicated in nociception and chronic pain including the voltage-gated sodium channel Nav1.7 and the voltage-gated potassium channels Kv1.1 and Kv1.2. We found that blocking Slc7a5 with intrathecal administration of the drug JPH203 alleviated allodynia in the spared nerve injury (SNI) rodent model of neuropathic pain. Western blot and immunohistochemistry studies revealed an increase in Slc7a5 protein levels in the spinal cord and DRGs of SNI mice compared to control mice. Using whole-cell current-clamp electrophysiology, we observed that JPH203 treatment reduced excitability of small-diameter (< 30 µm) DRG neurons from SNI mice, in agreement with its behavioral effects. Voltage-clamp recordings from JPH203-treated naïve rat DRGs identified an effect on tetrodotoxin-resistant (TTX-R) sodium currents. Altogether, these results demonstrate that Slc7a5 is dysregulated in chronic neuropathic pain and can be targeted to provide relief of hypersensitivity.

Targeting Amino Acid Metabolic Reprogramming via L-Type Amino Acid Transporter 1 (LAT1) for Endocrine-Resistant Breast Cancer.

The PI3K/Akt/mTOR pathway has been well known to interact with the estrogen receptor (ER)-pathway and to be also frequently upregulated in aromatase inhibitor (AI)-resistant breast cancer patients. Intracellular levels of free amino acids, especially leucine, regulate the mammalian target of rapamycin complex 1 (mTORC1) activation. L-type amino acid transporters such as LAT1 and LAT3 are associated with the uptake of essential amino acids. LAT1 expression could mediate leucine uptake, mTORC1 signaling, and cell proliferation. Therefore, in this study, we explored amino acid metabolism, including LAT1, in breast cancer and clarified the potential roles of LAT1 in the development of therapeutic resistance and the eventual clinical outcome of the patients. We evaluated LAT1 and LAT3 expression before and after neoadjuvant hormone therapy (NAH) and examined LAT1 function and expression in estrogen deprivation-resistant (EDR) breast carcinoma cell lines. Tumors tended to be in advanced stages in the cases whose LAT1 expression was high. LAT1 expression in the EDR cell lines was upregulated. JPH203, a selective LAT1 inhibitor, demonstrated inhibitory effects on cell proliferation in EDR cells. Hormone therapy changed the tumor microenvironment and resulted in metabolic reprogramming through inducing LAT1 expression. LAT1 expression then mediated leucine uptake, enhanced mTORC1 signaling, and eventually resulted in AI resistance. Therefore, LAT1 could be the potential therapeutic target in AI-resistant breast cancer patients.

Oncogenic KRAS mutations enhance amino acid uptake by colorectal cancer cells via the hippo signaling effector YAP1.

Oncogenic KRAS mutations develop unique metabolic dependencies on nutrients to support tumor metabolism and cell proliferation. In particular, KRAS mutant cancer cells exploit amino acids (AAs) such as glutamine and leucine, to accelerate energy metabolism, redox balance through glutathione synthesis and macromolecule biosynthesis. However, the identities of the amino acid transporters (AATs) that are prominently upregulated in KRAS mutant cancer cells, and the mechanism regulating their expression have not yet been systematically investigated. Here, we report that the majority of the KRAS mutant colorectal cancer (CRC) cells upregulate selected AATs (SLC7A5/LAT1, SLC38A2/SNAT2, and SLC1A5/ASCT2), which correlates with enhanced uptake of AAs such as glutamine and leucine. Consistently, knockdown of oncogenic KRAS downregulated the expression of AATs, thereby decreasing the levels of amino acids taken up by CRC cells. Moreover, overexpression of mutant KRAS upregulated the expression of AATs (SLC7A5/LAT1, SLC38A2/SNAT2, and SLC1A5/ASCT2) in KRAS wild-type CRC cells and mouse embryonic fibroblasts. In addition, we show that the YAP1 (Yes-associated protein 1) transcriptional coactivator accounts for increased expression of AATs and mTOR activation in KRAS mutant CRC cells. Specific knockdown of AATs by shRNAs or pharmacological blockage of AATs effectively inhibited AA uptake, mTOR activation, and cell proliferation. Collectively, we conclude that oncogenic KRAS mutations enhance the expression of AATs via the hippo effector YAP1, leading to mTOR activation and CRC cell proliferation.

Amino Acid Transporters on the Guard of Cell Genome and Epigenome.

Tumorigenesis is driven by metabolic reprogramming. Oncogenic mutations and epigenetic alterations that cause metabolic rewiring may also upregulate the reactive oxygen species (ROS). Precise regulation of the intracellular ROS levels is critical for tumor cell growth and survival. High ROS production leads to the damage of vital macromolecules, such as DNA, proteins, and lipids, causing genomic instability and further tumor evolution. One of the hallmarks of cancer metabolism is deregulated amino acid uptake. In fast-growing tumors, amino acids are not only the source of energy and building intermediates but also critical regulators of redox homeostasis. Amino acid uptake regulates the intracellular glutathione (GSH) levels, endoplasmic reticulum stress, unfolded protein response signaling, mTOR-mediated antioxidant defense, and epigenetic adaptations of tumor cells to oxidative stress. This review summarizes the role of amino acid transporters as the defender of tumor antioxidant system and genome integrity and discusses them as promising therapeutic targets and tumor imaging tools.

Wnt regulates amino acid transporter Slc7a5 and so constrains the integrated stress response in mouse embryos.

Amino acids are essential for cellular metabolism, and it is important to understand how nutrient supply is coordinated with changing energy requirements during embryogenesis. Here, we show that the amino acid transporter Slc7a5/Lat1 is highly expressed in tissues undergoing morphogenesis and that Slc7a5-null mouse embryos have profound neural and limb bud outgrowth defects. Slc7a5-null neural tissue exhibited aberrant mTORC1 activity and cell proliferation; transcriptomics, protein phosphorylation and apoptosis analyses further indicated induction of the integrated stress response as a potential cause of observed defects. The pattern of stress response gene expression induced in Slc7a5-null embryos was also detected at low level in wild-type embryos and identified stress vulnerability specifically in tissues undergoing morphogenesis. The Slc7a5-null phenotype is reminiscent of Wnt pathway mutants, and we show that Wnt/ß-catenin loss inhibits Slc7a5 expression and induces this stress response. Wnt signalling therefore normally supports the metabolic demands of morphogenesis and constrains cellular stress. Moreover, operation in the embryo of the integrated stress response, which is triggered by pathogen-mediated as well as metabolic stress, may provide a mechanistic explanation for a range of developmental defects.

Functional Polarity of Microvascular Brain Endothelial Cells Supported by Neurovascular Unit Computational Model of Large Neutral Amino Acid Homeostasis.

The homeostatic regulation of large neutral amino acid (LNAA) concentration in the brain interstitial fluid (ISF) is essential for proper brain function. LNAA passage into the brain is primarily mediated by the complex and dynamic interactions between various solute carrier (SLC) transporters expressed in the neurovascular unit (NVU), among which SLC7A5/LAT1 is considered to be the major contributor in microvascular brain endothelial cells (MBEC). The LAT1-mediated trans-endothelial transport of LNAAs, however, could not be characterized precisely by available in vitro and in vivo standard methods so far. To circumvent these limitations, we have incorporated published in vivo data of rat brain into a robust computational model of NVU-LNAA homeostasis, allowing us to evaluate hypotheses concerning LAT1-mediated trans-endothelial transport of LNAAs across the blood brain barrier (BBB). We show that accounting for functional polarity of MBECs with either asymmetric LAT1 distribution between membranes and/or intrinsic LAT1 asymmetry with low intraendothelial binding affinity is required to reproduce the experimentally measured brain ISF response to intraperitoneal (IP) L-tyrosine and L-phenylalanine injection. On the basis of these findings, we have also investigated the effect of IP administrated L-tyrosine and L-phenylalanine on the dynamics of LNAAs in MBECs, astrocytes and neurons. Finally, the computational model was shown to explain the trans-stimulation of LNAA uptake across the BBB observed upon ISF perfusion with a competitive LAT1 inhibitor.