Extracellular Matrix Remodeling Regulates Glucose Metabolism through TXNIP Destabilization.

The metabolic state of a cell is influenced by cell-extrinsic factors, including nutrient availability and growth factor signaling. Here, we present extracellular matrix (ECM) remodeling as another fundamental node of cell-extrinsic metabolic regulation. Unbiased analysis of glycolytic drivers identified the hyaluronan-mediated motility receptor as being among the most highly correlated with glycolysis in cancer. Confirming a mechanistic link between the ECM component hyaluronan and metabolism, treatment of cells and xenografts with hyaluronidase triggers a robust increase in glycolysis. This is largely achieved through rapid receptor tyrosine kinase-mediated induction of the mRNA decay factor ZFP36, which targets TXNIP transcripts for degradation. Because TXNIP promotes internalization of the glucose transporter GLUT1, its acute decline enriches GLUT1 at the plasma membrane. Functionally, induction of glycolysis by hyaluronidase is required for concomitant acceleration of cell migration. This interconnection between ECM remodeling and metabolism is exhibited in dynamic tissue states, including tumorigenesis and embryogenesis.

Inhibition of pyruvate oxidation as a versatile stimulator of the hair cycle in models of alopecia.

Hair follicle stem cells (HFSCs) are known to be responsible for the initiation of a new hair cycle, but typically remain quiescent for very long periods. In alopecia, or hair loss disorders, follicles can be refractory to activation for years or even permanently. Alopecia can be triggered by autoimmunity, age, chemotherapeutic treatment, stress, disrupted circadian rhythm or other environmental insults. We previously showed that hair follicle stem cells and the hair cycle can be manipulated by regulation of pyruvate entry into mitochondria for subsequent oxidation to fuel the TCA cycle in normal adult mice with typical hair cycling. Here, we present new data from our efforts to develop murine models of alopecia based on environmental triggers that have been shown to do the same in human skin. We found that inhibition of pyruvate transport into mitochondria can accelerate the hair cycle even during refractory hair cycling due to age, repeated chemotherapeutic treatment and stress. Hair cycle acceleration in these alopecia models led to the formation of histologically normal hair follicles within 30-40 days of treatment without any overt signs of toxicity or deleterious effects. Therefore, we propose inhibition of pyruvate entry into mitochondria as a versatile treatment strategy for alopecia in humans.

Hmga2 is dispensable for cutaneous squamous cell carcinoma.

Hmga2 functions as a chromatin-associated factor during development, but is not expressed in most adult tissues. Expression of Hmga2 in adult tissues has been associated with a variety of human cancers. Numerous studies have implicated Hmga2 in epithelial-to-mesenchymal transition (EMT) and cancer progression through gain of function studies, but it is unclear whether Hgma2 is necessary for EMT, tumor formation or tumor progression. We deleted Hmga2 in two mouse models of squamous cell carcinoma and found this gene to be dispensable. In fact, EMT, tumor initiation and progression all appeared to be mostly unaffected by the absence of Hmga2. Tumors lacking the ability to induce Hmga2 proceeded to initiate cutaneous spindle cell and squamous cell carcinomas with all the typical pathological and molecular hallmarks of these cancers.

Defining metabolic flexibility in hair follicle stem cell induced squamous cell carcinoma.

We previously showed that inhibition of glycolysis in cutaneous squamous cell carcinoma (SCC)-initiating cells had no effect on tumorigenesis, despite the perceived requirement of the Warburg effect, which was thought to drive carcinogenesis. Instead, these SCCs were metabolically flexible and sustained growth through glutaminolysis, another metabolic process frequently implicated to fuel tumorigenesis in various cancers. Here, we focused on glutaminolysis and genetically blocked this process through glutaminase (GLS) deletion in SCC cells of origin. Genetic deletion of GLS had little effect on tumorigenesis due to the up-regulated lactate consumption and utilization for the TCA cycle, providing further evidence of metabolic flexibility. We went on to show that posttranscriptional regulation of nutrient transporters appears to mediate metabolic flexibility in this SCC model. To define the limits of this flexibility, we genetically blocked both glycolysis and glutaminolysis simultaneously and found the abrogation of both of these carbon utilization pathways was enough to prevent both papilloma and frank carcinoma.

Development of Novel Mitochondrial Pyruvate Carrier Inhibitors to Treat Hair Loss.

Herein, we report the synthesis and evaluation of novel analogues of UK-5099 both in vitro and in vivo for the development of mitochondrial pyruvate carrier (MPC) inhibitors to treat hair loss. A comprehensive understanding of the structure-activity relationship was obtained by varying four positions of the hit compound, namely, the alkyl group on the N1 position, substituents on the indole core, various aromatic and heteroaromatic core structures, and various Michael acceptors. The major discovery was that the inhibitors with a 3,5-bis(trifluoromethyl)benzyl group at the N1 position were shown to have much better activity than JXL001 (UK-5099) to increase cellular lactate production. Additionally, analogue JXL069, possessing a 7-azaindole heterocycle, was also shown to have significant MPC inhibition activity, which further increases the chemical space for drug design. Finally, more than 10 analogues were tested on shaved mice by topical treatment and promoted obvious hair growth on mice.

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

Lactate dehydrogenase activity drives hair follicle stem cell activation.

Although normally dormant, hair follicle stem cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier 1 (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allows them to remain dormant and yet quickly respond to appropriate proliferative stimuli.

Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism.

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells.

Exploiting Mouse Models to Study Ras-Induced Cutaneous Squamous Cell Carcinoma.

Recently developed methods have allowed for the delivery of cancer-causing genetic mutations to particular cell types in the epidermis in an inducible fashion. These methods have allowed for sophisticated explorations on the cellular and molecular origins of squamous cell carcinoma due to oncogenic mutations in Ras. These experiments have provided insights into whether cancer is initiated by stem or more specified cells under various conditions, and have highlighted the ability of particular genetic hits to serve as tumor initiators or promoters. Here we provide a summary of data from our lab and others that demonstrate the ability of hair follicle stem cells to serve as cancer cells of origin, and the ability of various molecular players to drive heterogeneity of tumor cell types. A synthesis of these studies potentially could provide unique insights into the process by which Ras can initiate squamous cell carcinoma in human patients and could eventually inform treatment strategies.