Knockout model reveals the role of Nischarin in mammary gland development, breast tumorigenesis and response to metformin treatment.

Previously, our lab discovered the protein Nischarin and uncovered its role in regulating cell migration and invasion via its interactions with several proteins. We subsequently described a role for Nischarin in breast cancer, in which it is frequently underexpressed. To characterize Nischarin's role in breast tumorigenesis and mammary gland development more completely, we deleted a critical region of the Nisch gene (exons 7-10) from the mouse genome and observed the effects. Mammary glands in mutant animals showed delayed terminal end bud formation but did not develop breast tumors spontaneously. Therefore, we interbred the animals with transgenic mice expressing the mouse mammary tumor virus-polyoma middle T-antigen (MMTV-PyMT) oncogene. The MMTV-PyMT mammary glands lacking Nischarin showed increased hyperplasia compared to wild-type animal tissues. Furthermore, we observed significantly increased tumor growth and metastasis in Nischarin mutant animals. Surprisingly, Nischarin deletion decreased activity of AMPK and subsequently its downstream effectors. Given this finding, we treated these animals with metformin, which enhances AMPK activity. Here, we show for the first time, metformin activates AMPK signaling and inhibits tumor growth of Nischarin lacking PyMT tumors suggesting a potential use for metformin as a cancer therapeutic, particularly in the case of Nischarin-deficient breast cancers.

Nischarin inhibition alters energy metabolism by activating AMP-activated protein kinase.

Nischarin (Nisch) is a key protein functioning as a molecular scaffold and thereby hosting interactions with several protein partners. To explore the physiological importance of Nisch, here we generated Nisch loss-of-function mutant mice and analyzed their metabolic phenotype. Nisch-mutant embryos exhibited delayed development, characterized by small size and attenuated weight gain. We uncovered the reason for this phenotype by showing that Nisch binds to and inhibits the activity of AMP-activated protein kinase (AMPK), which regulates energy homeostasis by suppressing anabolic and activating catabolic processes. The Nisch mutations enhanced AMPK activation and inhibited mechanistic target of rapamycin signaling in mouse embryonic fibroblasts as well as in muscle and liver tissues of mutant mice. Nisch-mutant mice also exhibited increased rates of glucose oxidation with increased energy expenditure, despite reduced overall food intake. Moreover, the Nisch-mutant mice had reduced expression of liver markers of gluconeogenesis associated with increased glucose tolerance. As a result, these mice displayed decreased growth and body weight. Taken together, our results indicate that Nisch is an important AMPK inhibitor and a critical regulator of energy homeostasis, including lipid and glucose metabolism.

Suppression of PDHX by microRNA-27b deregulates cell metabolism and promotes growth in breast cancer.

BACKGROUND: The disruption of normal gene regulation due to microRNA dysfunction is a common event in cancer pathogenesis. MicroRNA-27b is an example of an oncogenic miRNA, and it is frequently upregulated in breast cancer. MicroRNAs have been found to deregulate tumor metabolism, which typically manifests as heightened cellular glucose uptake in consort with increased flux through glycolysis, followed by the preferential conversion of glycolytic pyruvate into lactate (a phenomenon known as the Warburg Effect). Pyruvate Dehydrogenase, an enzyme complex linking glycolysis with downstream oxidative metabolism, represents a key location where regulation of metabolism occurs; PDHX is a key structural component of this complex and is essential for its function. METHODS: We sought to characterize the role of miR-27b in breast cancer by identifying novel transcripts under its control. We began by utilizing luciferase, RNA, and protein assays to establish PDHX as a novel target of miR-27b. We then tested whether miR-27b could alter metabolism using several metabolite assay kits and performed a seahorse analysis. We also examined how the altered metabolism might affect cell proliferation. Lastly, we confirmed the relevance of our findings in human breast tumor samples. RESULTS: Our data indicate that Pyruvate Dehydrogenase Protein X is a credible target of miR-27b in breast cancer. Mechanistically, by suppressing PDHX, miR-27b altered levels of pyruvate, lactate and citrate, as well as reducing mitochondrial oxidation and promoting extracellular acidification. These changes corresponded with an increased capacity for cell proliferation. In human breast tumor samples, PDHX expression was deficient, and low levels of PDHX were associated with reduced patient survival. CONCLUSIONS: MicroRNA-27b targets PDHX, resulting in an altered metabolic configuration that is better suited to fuel biosynthetic processes and cell proliferation, thereby promoting breast cancer progression.

Kratom-Pharmacology, Clinical Implications, and Outlook: A Comprehensive Review.

Kratom, or Mitragyna, is a tropical plant indigenous to Southeast Asia, with unique pharmacological properties. It is commonly consumed by preparing the leaves into decoction or tea, or by grinding them into a powder. Recent evidence has revealed that kratom has physiological effects similar to opioids, including pain relief and euphoria, as well as stimulant properties, which together raise potential concern for dependence and addiction. Moreover, growing evidence suggests that the prevalence of kratom use is increasing in many parts of the world, raising important considerations for healthcare providers. This manuscript will discuss the most current epidemiology, pharmacology, toxicity, and management related to kratom, while seeking to provide a contemporary perspective on the issue and its role in the greater context of the opioid epidemic.

Expression of long noncoding RNA MALAT1 correlates with increased levels of Nischarin and inhibits oncogenic cell functions in breast cancer.

Malat1 is a long noncoding RNA with a wide array of functions, including roles in regulating cancer cell migration and metastasis. However, the nature of its involvement in control of these oncogenic processes is incompletely understood. In the present study, we investigate the role of Malat1 and the effects of Malat1 KO in a breast cancer cell model. Our selection of Malat1 as the subject of inquiry followed initial screening experiments seeking to identify lncRNAs which are altered in the presence or absence of Nischarin, a gene of interest previously discovered by our lab. Nischarin is a well characterized tumor suppressor protein and actively represses cell proliferation, migration, and invasion in breast cancer. Our microarray screen for lncRNAs revealed multiple lncRNAs to be significantly elevated in cells ectopically expressing Nischarin compared to control cancer cells, which have only marginal Nisch expression. Using these cells, we assess how the link between Nischarin and Malat1 affects cancer cell function, finding that Malat1 confers an inhibitory effect on cell growth and migration which is lost following Malat1 KO, but in a Nisch-dependent context. Specifically, Malat1 KO in the background of low Nischarin expression had a limited effect on cell functions, while Malat1 KO in cells with high levels of Nischarin led to significant increases in cell proliferation and migration. In summary, this project provides further clarity concerning the function of Malat1, specifically in breast cancer, while also indicating that the Nischarin expression context is an important factor in the determining how Malat1 activity is governed in breast cancer.

MicroRNA and Breast Cancer: Understanding Pathogenesis, Improving Management.

The advent of the microRNAs in the early 1990s has proven to be a tremendously significant development within the purview of gene regulation. They participate in the regulation of a broad assembly of processes vital to proper cell function and the perturbation of these pathways following alteration of miRNA expression is strongly believed to contribute to the pathogenesis of cancer. This review provides a comprehensive overview of the miRNAs that have to date been well-characterized in the context of human breast neoplasia. Detailed discussion will center around their role in tumor initiation and progression, control of epithelial-mesenchymal transition (EMT), cancer stem cell formation, use as biomarkers in tissues and circulation, as well as their role in cancer treatment. In addition, attention will be given to topics which remain underexplored, such as miRNA control of cancer cell metabolism and the genomic/epigenetic origins underlying the preliminary disruption of miRNA expression in disease. This review will also address and attempt to resolve instances where discordant, inter-study findings have been reported (examples of which are replete in the literature) while also identifying bottlenecks hampering progress in miRNA research and other challenges that confront this fledgling but promising field of biomedical research.

Restraint of the G2/M transition by the SR/RRM family mRNA shuttling binding protein SNXAHRB1 in Aspergillus nidulans.

Control of the eukaryotic G2/M transition by CDC2/CYCLINB is tightly regulated by protein-protein interactions, protein phosphorylations, and nuclear localization of CDC2/CYCLINB. We previously reported a screen, in Aspergillus nidulans, for extragenic suppressors of nimX2(cdc2) that resulted in the identification of the cold-sensitive snxA1 mutation. We demonstrate here that snxA1 suppresses defects in regulators of the CDK1 mitotic induction pathway, including nimX2(cdc) (2), nimE6(cyclinB), and nimT23(cdc) (25), but does not suppress G2-arresting nimA1/nimA5 mutations, the S-arresting nimE10(cyclinB) mutation, or three other G1/S phase mutations. snxA encodes the A. nidulans homolog of Saccharomyces cerevisiae Hrb1/Gbp2; nonessential shuttling messenger RNA (mRNA)-binding proteins belonging to the serine-arginine-rich (SR) and RNA recognition motif (RRM) protein family; and human heterogeneous ribonucleoprotein-M, a spliceosomal component involved in pre-mRNA processing and alternative splicing. snxA(Hrb) (1) is nonessential, its deletion phenocopies the snxA1 mutation, and its overexpression rescues snxA1 and ΔsnxA mutant phenotypes. snxA1 and a second allele isolated in this study, snxA2, are hypomorphic mutations that result from decreased transcript and protein levels, suggesting that snxA acts normally to restrain cell cycle progression. SNXA(HRB1) is predominantly nuclear, but is not retained in the nucleus during the partially closed mitosis of A. nidulans. We show that the snxA1 mutation does not suppress nimX2 by altering NIMX2(CDC2)/NIME(CYCLINB) kinase activity and that snxA1 or ΔsnxA alter localization patterns of NIME(CYCLINB) at the restrictive temperatures for snxA1 and nimX2. Together, these findings suggest a novel and previously unreported role of an SR/RRM family protein in cell cycle regulation, specifically in control of the CDK1 mitotic induction pathway.