TFEB deficiency attenuates mitochondrial degradation upon brown adipose tissue whitening at thermoneutrality.

OBJECTIVE: Brown adipose tissue (BAT) thermogenesis offers the potential to improve metabolic health in mice and humans. However, humans predominantly live under thermoneutral conditions, leading to BAT whitening, a reduction in BAT mitochondrial content and metabolic activity. Recent studies have established mitophagy as a major driver of mitochondrial degradation in the whitening of thermogenic brite/beige adipocytes, yet the pathways mediating mitochondrial breakdown in whitening of classical BAT remain largely elusive. The transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy belonging to the MiT family of transcription factors, is the only member of this family that is upregulated during whitening, pointing toward a role of TFEB in whitening-associated mitochondrial breakdown. METHODS: We generated brown adipocyte-specific TFEB knockout mice, and induced BAT whitening by thermoneutral housing. We characterized gene and protein expression patterns, BAT metabolic activity, systemic metabolism, and mitochondrial localization using in vivo and in vitro approaches. RESULTS: Under low thermogenic activation conditions, deletion of TFEB preserves mitochondrial mass independently of mitochondriogenesis in BAT and primary brown adipocytes. However, this does not translate into elevated thermogenic capacity or protection from diet-induced obesity. Autophagosomal/lysosomal marker levels are altered in TFEB-deficient BAT and primary adipocytes, and lysosomal markers co-localize and co-purify with mitochondria in TFEB-deficient BAT, indicating trapping of mitochondria in late stages of mitophagy. CONCLUSION: We identify TFEB as a driver of BAT whitening, mediating mitochondrial degradation via the autophagosomal and lysosomal machinery. This study provides proof of concept that interfering with the mitochondrial degradation machinery can increase mitochondrial mass in classical BAT under human-relevant conditions. However, it must be considered that interfering with autophagy may result in accumulation of non-functional mitochondria. Future studies targeting earlier steps of mitophagy or target recognition are therefore warranted.

Activation of TFEB-mediated autophagy by trehalose attenuates mitochondrial dysfunction in cisplatin-induced acute kidney injury.

Aims: Cisplatin, an anticancer drug, always leads to nephrotoxicity by causing mitochondrial dysfunction. As a major mechanism for cellular self-degradation, autophagy has been proven to protect against cisplatin-induced acute kidney injury (AKI). Based on the activation of autophagy induced by trehalose, we aimed to investigate the nephroprotective effects of trehalose on cisplatin-induced AKI and its underlying mechanisms. Results: Due to the activation of autophagy, mitochondrial dysfunction (mitochondrial fragmentation, depolarization, reactive oxygen species (ROS), and reduced ATP generation) and apoptosis induced by cisplatin were markedly inhibited in trehalose-treated HK2 cells in vitro. Based on the transcriptional regulation role of transcription factor EB (TFEB) in autophagy and lysosome, we characterized trehalose-induced nuclear translocation of TFEB. Furthermore, consistent with trehalose treatment, overexpression of TFEB inhibited cell injury induced by cisplatin. However, the protective effects of trehalose were largely abrogated in tfeb-knockdown cells. In vivo, cisplatin injection resulted in severe kidney dysfunction and histological damage in mice. Trehalose administration activated TFEB-mediated autophagy, alleviated mitochondrial dysfunction and kidney injury in AKI mice. Innovation and conclusion: Our data suggest that trehalose treatment preserves mitochondria function via activation of TFEB-mediated autophagy and attenuates cisplatin-induced kidney injury.

Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan.

Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca2+ dependent manner. Cytosolic Ca2+ concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca2+ increase, thereby suppressing induction of autophagy.

Gold nanoclusters elicit homeostatic perturbations in glioblastoma cells and adaptive changes of lysosomes.

Unique physicochemical features place gold nanoclusters at the forefront of nanotechnology for biological and biomedical applications. To date, information on the interactions of gold nanoclusters with biological macromolecules is limited and restricts their use in living cells. Methods: Our multidisciplinary study begins to fill the current knowledge gap by focusing on lysosomes and associated biological pathways in U251N human glioblastoma cells. We concentrated on lysosomes, because they are the intracellular destination for many nanoparticles, regulate cellular homeostasis and control cell survival. Results: Quantitative data presented here show that gold nanoclusters (with 15 and 25 gold atoms), surface-modified with glutathione or PEG, did not diminish cell viability at concentrations ≤1 µM. However, even at sublethal concentrations, gold nanoclusters modulated the abundance, positioning, pH and enzymatic activities of lysosomes. Gold nanoclusters also affected other aspects of cellular homeostasis. Specifically, they stimulated the transient nuclear accumulation of TFEB and Nrf2, transcription factors that promote lysosome biogenesis and stress responses. Moreover, gold nanoclusters also altered the formation of protein aggregates in the cytoplasm. The cellular responses elicited by gold nanoclusters were largely reversible within a 24-hour period. Conclusions: Taken together, this study explores the subcellular and molecular effects induced by gold nanoclusters and shows their effectiveness to regulate lysosome biology. Our results indicate that gold nanoclusters cause homeostatic perturbations without marked cell loss. Notably, cells adapt to the challenge inflicted by gold nanoclusters. These new insights provide a framework for the further development of gold nanocluster-based applications in biological sciences.

SPZ1 promotes deregulation of Bim to boost apoptosis resistance in colorectal cancer.

Colorectal cancer (CRC) is the third most common malignancies in adults. Similar to other solid tumors, CRC cells show increased proliferation and suppressed apoptosis during the development and progression of the disease. Previous studies have shown that a novel tumor oncogene, spermatogenic basic helix-loop-helix transcription factor zip 1 (SPZ1), can promote proliferation. However, it is unclear whether SPZ1 plays a role in suppressing apoptosis, and the molecular mechanism behind SPZ1's suppression of apoptosis in CRC remains unclear. Here, we found that silencing endogenous SPZ1 inhibits cell growth and induces apoptosis, and overexpression of SPZ1 promotes cell growth. These findings were corroborated by in vitro and in vivo studies. Interestingly, SPZ1 overexpressing cells were resistant to 5-fluorouracil, a drug commonly used to treat cancer. Moreover, knocking down SPZ1 led to the activation of caspase through the deregulation of Bim by ERK1/2, we found that CRC tissues had significantly higher SPZ1 and lower Bim expression, and SPZ1HBimL were associated with advanced clinical stage of CRC. Collectively, our findings demonstrate that SPZ1 contributes to tumor progression by limiting apoptosis. SPZ1 reduces apoptosis by altering the stability of Bim, suggesting SPZ1 may serve as a biomarker and therapeutic target for CRC.

Lysosomal Fusion: An Efficient Mechanism Increasing Their Sequestration Capacity for Weak Base Drugs without Apparent Lysosomal Biogenesis.

Lysosomal sequestration of anticancer therapeutics lowers their cytotoxic potential, reduces drug availability at target sites, and contributes to cancer resistance. Only recently has it been shown that lysosomal sequestration of weak base drugs induces lysosomal biogenesis mediated by activation of transcription factor EB (TFEB) which, in turn, enhances their accumulation capacity, thereby increasing resistance to these drugs. Here, we addressed the question of whether lysosomal biogenesis is the only mechanism that increases lysosomal sequestration capacity. We found that lysosomal sequestration of some tyrosine kinase inhibitors (TKIs), gefitinib (GF) and imatinib (IM), induced expansion of the lysosomal compartment. However, an expression analysis of lysosomal genes, including lysosome-associated membrane proteins 1, 2 (LAMP1, LAMP2), vacuolar ATPase subunit B2 (ATP6V1B2), acid phosphatase (ACP), and galactosidase beta (GLB) controlled by TFEB, did not reveal increased expression. Instead, we found that both studied TKIs, GF and IM, induced lysosomal fusion which was dependent on nicotinic acid adenine dinucleotide phosphate (NAADP) mediated Ca2+signaling. A theoretical analysis revealed that lysosomal fusion is sufficient to explain the enlargement of lysosomal sequestration capacity. In conclusion, we demonstrated that extracellular TKIs, GF and IM, induced NAADP/Ca2+ mediated lysosomal fusion, leading to enlargement of the lysosomal compartment with significantly increased sequestration capacity for these drugs without apparent lysosomal biogenesis.

Nutrient-sensitive transcription factors TFEB and TFE3 couple autophagy and metabolism to the peripheral clock.

Autophagy and energy metabolism are known to follow a circadian pattern. However, it is unclear whether autophagy and the circadian clock are coordinated by common control mechanisms. Here, we show that the oscillation of autophagy genes is dependent on the nutrient-sensitive activation of TFEB and TFE3, key regulators of autophagy, lysosomal biogenesis, and cell homeostasis. TFEB and TFE3 display a circadian activation over the 24-h cycle and are responsible for the rhythmic induction of genes involved in autophagy during the light phase. Genetic ablation of TFEB and TFE3 in mice results in deregulated autophagy over the diurnal cycle and altered gene expression causing abnormal circadian wheel-running behavior. In addition, TFEB and TFE3 directly regulate the expression of Rev-erbα (Nr1d1), a transcriptional repressor component of the core clock machinery also involved in the regulation of whole-body metabolism and autophagy. Comparative analysis of the cistromes of TFEB/TFE3 and REV-ERBα showed an extensive overlap of their binding sites, particularly in genes involved in autophagy and metabolic functions. These data reveal a direct link between nutrient and clock-dependent regulation of gene expression shedding a new light on the crosstalk between autophagy, metabolism, and circadian cycles.

Inhibition of TFEB oligomerization by co-treatment of melatonin with vorinostat promotes the therapeutic sensitivity in glioblastoma and glioma stem cells.

Glioblastoma (GBM) is the most aggressive malignant glioma and most lethal form of human brain cancer (Clin J Oncol Nurs. 2016;20:S2). GBM is also one of the most expensive and difficult cancers to treat by the surgical resection, local radiotherapy, and temozolomide (TMZ) and still remains an incurable disease. Oncomine platform analysis and Gene Expression Profiling Interactive Analysis (GEPIA) show that the expression of transcription factor EB (TFEB) was significantly increased in GBMs and in GBM patients above stage IV. TFEB requires the oligomerization and localization to regulate transcription in the nucleus. Also, the expression and oligomerization of TFEB proteins contribute to the resistance of GBM cells to conventional chemotherapeutic agents such as TMZ. Thus, we investigated whether the combination of vorinostat and melatonin could overcome the effects of TFEB and induce apoptosis in GBM cells and glioma cancer stem cells (GSCs). The downregulation of TFEB and oligomerization by vorinostat and melatonin increased the expression of apoptosis-related genes and activated the apoptotic cell death process. Significantly, the inhibition of TFEB expression dramatically decreased GSC tumor-sphere formation and size. The inhibitory effect of co-treatment resulted in decreased proliferation of GSCs and induced the expression of cleaved PARP and p-γH2AX. Taken together, our results definitely demonstrate that TFEB expression contributes to enhanced resistance of GBMs to chemotherapy and that vorinostat- and melatonin-activated apoptosis signaling in GBM cells by inhibiting TFEB expression and oligomerization, suggesting that co-treatment of vorinostat and melatonin may be an effective therapeutic strategy for human brain cancers.

Alantolactone induces apoptosis and improves chemosensitivity of pancreatic cancer cells by impairment of autophagy-lysosome pathway via targeting TFEB.

The lysosome is emerging as a central regulator of the autophagic process, which plays a critical role in tumor growth and chemoresistance. Alantolactone, which is a natural compound produced by Inula helenium, has been shown to induce apoptosis in numerous cancer types. However, the mechanism by which alantolactone regulates apoptosis is still poorly understood. In this work, we observed that alantolactone caused the accumulation of autophagosomes due to impaired autophagic degradation and substantially inhibited the activity and expression of CTSB/CTSD proteins that when depleted caused lysosomal dysfunction. Furthermore, we found that alantolactone inhibited the proliferation of pancreatic cancer cells in vitro and in vivo and enhanced the chemosensitivity of pancreatic cancer cells to oxaliplatin. In addition, a reduction in TFEB levels was a critical event in the apoptosis and cell death caused by alantolactone. Our data demonstrated that alantolactone, which impaired autophagic degradation, was a pharmacological inhibitor of autophagy in pancreatic cancer cells and markedly enhanced the chemosensitivity of pancreatic cancer cells to oxaliplatin.

TFEB and trehalose drive the macrophage autophagy-lysosome system to protect against atherosclerosis.

In the atherosclerotic plaque, macrophages are the key catabolic workhorse responsible for clearing lipid and dead cell debris. To survive the highly proinflammatory and lipotoxic plaque environment, macrophages must adopt strategies for maintaining tight homeostasis and self-renewal. Macroautophagy/autophagy is a pro-survival cellular pathway wherein damaged or excess cellular cargoes are encapsulated by a double-membrane compartment and delivered to the lysosome for hydrolysis. Previously, macrophage-specific autophagy deficiency has been shown to be atherogenic through several complementary mechanisms including hyperactivation of the inflammasome, defective efferocytosis, accumulation of cytotoxic protein aggregates, and impaired lipid degradation. Conversely, in a recent study we hypothesized that enhancing the macrophage autophagy-lysosomal system through genetic or pharmacological means could protect against atherosclerosis. We demonstrated that TFEB, a transcription factor master regulator of autophagy and lysosome biogenesis, coordinately enhances the function of this system to reduce atherosclerotic plaque burden. Further, we characterized the disaccharide trehalose as a novel inducer of TFEB with similar atheroprotective effects. Overall, these findings mechanistically interrogate the importance and therapeutic promise of a functional autophagy-lysosome degradation system in plaque macrophage biology.

6-Hydroxydopamine induces autophagic flux dysfunction by impairing transcription factor EB activation and lysosomal function in dopaminergic neurons and SH-SY5Y cells.

Autophagy deregulation has been implicated in Parkinson's disease (PD), yet the role of autophagy in neuronal survival remains controversial. In this study, we comprehensively investigated the time-course of autophagy-related markers in 6-OHDA-induced Parkinsonian rat models and assessed its effect on the state of autophagic flux both in vivo and in vitro. We observed an early activation of autophagy followed by autophagic flux impairment, which was confirmed with autophagy inhibitor chloroquine in vivo and Ad-GFP-mCherry-LC3-infected SH-SY5Y cells in vitro. In addition, 6-OHDA not only remarkably reduced the expression level of lysosome-associated membrane protein 1 (Lamp1), but also impaired the hydrolase activities of lysosomal proteases. Transcription factor EB (TFEB), a key transcription factor controlling lysosome biogenesis, was also significantly downregulated by 6-OHDA and its nuclear translocation was inhibited as well, which could account for the impaired lysosomal function. Promoting lysosome biogenesis through TFEB overexpression could protect SH-SY5Y cells against 6-OHDA-induced neurotoxicity. The above findings demonstrated that autophagic flux dysfunction was closely associated with 6-OHDA-induced neurotoxicity and highlighted the importance of functional lysosomes and homeostatic autophagic flux in developing therapeutic agents for PD.

Protein coingestion with alcohol following strenuous exercise attenuates alcohol-induced intramyocellular apoptosis and inhibition of autophagy.

Alcohol ingestion decreases postexercise rates of muscle protein synthesis, but the mechanism(s) (e.g., increased protein breakdown) underlying this observation is unknown. Autophagy is an intracellular "recycling" system required for homeostatic substrate and organelle turnover; its dysregulation may provoke apoptosis and lead to muscle atrophy. We investigated the acute effects of alcohol ingestion on autophagic cell signaling responses to a bout of concurrent (combined resistance- and endurance-based) exercise. In a randomized crossover design, eight physically active males completed three experimental trials of concurrent exercise with either postexercise ingestion of alcohol and carbohydrate (12 ± 2 standard drinks; ALC-CHO), energy-matched alcohol and protein (ALC-PRO), or protein (PRO) only. Muscle biopsies were taken at rest and 2 and 8 h postexercise. Select autophagy-related gene (Atg) proteins decreased compared with rest with ALC-CHO (P < 0.05) but not ALC-PRO. There were parallel increases (P < 0.05) in p62 and PINK1 commensurate with a reduction in BNIP3 content, indicating a diminished capacity for mitochondria-specific autophagy (mitophagy) when alcohol and carbohydrate were coingested. DNA fragmentation increased in both alcohol conditions (P < 0.05); however, nuclear AIF accumulation preceded this apoptotic response with ALC-CHO only (P < 0.05). In contrast, increases in the nuclear content of p53, TFEB, and PGC-1α in ALC-PRO were accompanied by markers of mitochondrial biogenesis at the transcriptional (Tfam, SCO2, and NRF-1) and translational (COX-IV, ATPAF1, and VDAC1) level (P < 0.05). We conclude that alcohol ingestion following exercise triggers apoptosis, whereas the anabolic properties of protein coingestion may stimulate mitochondrial biogenesis to protect cellular homeostasis.

Novel Epidermal Growth Factor Receptor Inhibitor Attenuates Angiotensin II-Induced Kidney Fibrosis.

Chronic activation of renin-angiotensin system (RAS) greatly contributes to renal fibrosis and accelerates the progression of chronic kidney disease; however, the underlying molecular mechanism is poorly understood. Angiotensin II (Ang II), the central component of RAS, is a key regulator of renal fibrogenic destruction. Here we show that epidermal growth factor receptor (EGFR) plays an important role in Ang II-induced renal fibrosis. Inhibition of EGFR activation by novel small molecules or by short hairpin RNA knockdown in Ang II-treated SV40 mesangial cells in vitro suppresses protein kinase B and extracellular signal-related kinase signaling pathways and transforming growth factor-ß/Sma- and Mad-related protein activation, and abolishes the accumulation of fibrotic markers such as connective tissue growth factor, collagen IV. The transactivation of EGFR by Ang II in SV40 cells depends on the phosphorylation of proto-oncogene tyrosine-protein kinase Src (c-Src) kinase. Further validation in vivo demonstrates that EGFR small molecule inhibitor successfully attenuates renal fibrosis and kidney dysfunction in a mouse model induced by Ang II infusion. These findings indicate a crucial role of EGFR in Ang II-dependent renal deterioration, and reveal EGFR inhibition as a new therapeutic strategy for preventing progression of chronic renal diseases.

Androgen receptor (AR) suppresses normal human prostate epithelial cell proliferation via AR/ß-catenin/TCF-4 complex inhibition of c-MYC transcription.

INTRODUCTION: Physiologic testosterone continuously stimulates prostate stromal cell secretion of paracrine growth factors (PGFs), which if unopposed would induce hyperplastic overgrowth of normal prostate epithelial cells (PrECs). METHODS: Lentiviral shRNA stable knock down of c-MYC, ß-catenin, or TCF-4 completely inhibits normal (i.e., non-transformed) human PrECs growth. c-MYC enhancer driven reporter expression and growth is inhibited by two chemically distinct molecules, which prevent ß-catenin signaling either by blocking TCF-4 binding (i.e., toxoflavin) or by stimulating degradation (i.e., AVX939). Recombinant DKK1 protein at a dose, which inhibits activation of canonical Wnt signaling does not inhibit PrEC growth. Nuclear ß-catenin translocation and PrEC growth is prevented by both lack of PGFs or Akt inhibitor-I. Growth inhibition induced by lack of PGFs, toxoflavin, or Akt inhibitor-I is overcome by constitutive c-MYC transcription. RESULTS: In the presence of continuous PGF signaling, PrEC hyperplasia is prevented by androgen binding to AR suppressing c-MYC transcription, resulting in G0 arrest/terminal differentiation independent of Rb, p21, p27, FoxP3, or down regulation of growth factors receptors and instead involves androgen-induced formation of AR/ß-catenin/TCF-4 complexes, which suppress c-MYC transcription. Such suppression does not occur when AR is mutated in its zinc-finger binding domain. DISCUSSION: Proliferation of non-transformed human PrECs is dependent upon c-MYC transcription via formation/binding of ß-catenin/TCF-4 complexes at both 5' and 3' c-MYC enhancers stimulated by Wnt-independent, PGF induced Akt signaling. In the presence of continuous PGF signaling, PrEC hyperplasia is prevented by androgen-induced formation of AR/ß-catenin/TCF-4 complexes, which retains binding to 3' c-MYC enhancer, but now suppresses c-MYC transcription.

Activation of the transcription factor carbohydrate-responsive element-binding protein by glucose leads to increased pancreatic beta cell differentiation in rats.

AIMS/HYPOTHESIS: Pancreatic cell development is a tightly controlled process. Although information is available regarding the mesodermal signals that control pancreatic development, little is known about the role of environmental factors such as nutrients, including glucose, on pancreatic development. We previously showed that glucose and its metabolism through the hexosamine biosynthesis pathway (HBP) promote pancreatic endocrine cell differentiation. Here, we analysed the role of the transcription factor carbohydrate-responsive element-binding protein (ChREBP) in this process. This transcription factor is activated by glucose, and has been recently described as a target of the HBP. METHODS: We used an in vitro bioassay in which pancreatic endocrine and exocrine cells develop from rat embryonic pancreas in a way that mimics in vivo pancreatic development. Using this model, gain-of-function and loss-of-function experiments were undertaken. RESULTS: ChREBP was produced in the endocrine lineage during pancreatic development, its abundance increasing with differentiation. When rat embryonic pancreases were cultured in the presence of glucose or xylitol, the production of ChREBP targets was induced. Concomitantly, beta cell differentiation was enhanced. On the other hand, when embryonic pancreases were cultured with inhibitors decreasing ChREBP activity or an adenovirus producing a dominant-negative ChREBP, beta cell differentiation was reduced, indicating that ChREBP activity was necessary for proper beta cell differentiation. Interestingly, adenovirus producing a dominant-negative ChREBP also reduced the positive effect of N-acetylglucosamine, a substrate of the HBP acting on beta cell differentiation. CONCLUSIONS/INTERPRETATION: Our work supports the idea that glucose, through the transcription factor ChREBP, controls beta cell differentiation from pancreatic progenitors.

Cadmium induces Wnt signaling to upregulate proliferation and survival genes in sub-confluent kidney proximal tubule cells.

BACKGROUND: The class 1 carcinogen cadmium (Cd2+) disrupts the E-cadherin/beta-catenin complex of epithelial adherens junctions (AJs) and causes renal cancer. Deregulation of E-cadherin adhesion and changes in Wnt/beta-catenin signaling are known to contribute to carcinogenesis. RESULTS: We investigated Wnt signaling after Cd2+-induced E-cadherin disruption in sub-confluent cultured kidney proximal tubule cells (PTC). Cd2+ (25 microM, 3-9 h) caused nuclear translocation of beta-catenin and triggered a Wnt response measured by TOPflash reporter assays. Cd2+ reduced the interaction of beta-catenin with AJ components (E-cadherin, alpha-catenin) and increased binding to the transcription factor TCF4 of the Wnt pathway, which was upregulated and translocated to the nucleus. While Wnt target genes (c-Myc, cyclin D1 and ABCB1) were up-regulated by Cd2+, electromobility shift assays showed increased TCF4 binding to cyclin D1 and ABCB1 promoter sequences with Cd2+. Overexpression of wild-type and mutant TCF4 confirmed Cd2+-induced Wnt signaling. Wnt signaling elicited by Cd2+ was not observed in confluent non-proliferating cells, which showed increased E-cadherin expression. Overexpression of E-cadherin reduced Wnt signaling, PTC proliferation and Cd2+ toxicity. Cd2+ also induced reactive oxygen species dependent expression of the pro-apoptotic ER stress marker and Wnt suppressor CHOP/GADD153 which, however, did not abolish Wnt response and cell viability. CONCLUSIONS: Cd2+ induces Wnt signaling in PTC. Hence, Cd2+ may facilitate carcinogenesis of PTC by promoting Wnt pathway-mediated proliferation and survival of pre-neoplastic cells.

Insulin alters the expression of components of the Wnt signaling pathway including TCF-4 in the intestinal cells.

BACKGROUND: Epidemiological and experimental evidence that support the correlation between Type 2 diabetes mellitus (T2D) and increased risks of colorectal cancer formation have led us to hypothesize the existence of molecular crosstalk between insulin and canonical Wnt signaling pathways. Insulin was shown to stimulate Wnt target gene expression, utilizing the effector of the Wnt signaling pathway. Whether insulin affects expression of components of Wnt pathway has not been extensively examined. METHODS: cDNA microarray was utilized to assess the effect of insulin on gene expression profile in the rat intestinal non-cancer IEC-6 cell line, followed by real-time RT-PCR, Western blotting and reporter gene analyses in intestinal cancer and non-cancer cells. RESULTS: Insulin was shown to alter the expression of a dozen of Wnt pathway related genes including TCF-4 (=TCF7L2) and frizzled- (Fzd-4). The stimulatory effect of insulin on TCF-4 expression was then confirmed by real-time RT-PCR, Western blotting and luciferase reporter analyses, while the activation on Fzd-4 was confirmed by real-time PCR. GENERAL SIGNIFICANCE: Our observations suggest that insulin may crosstalk with the Wnt signaling pathway in a multi-level fashion, involving insulin regulation of the expression of Wnt target genes, a Wnt receptor, as well as mediators of the Wnt signaling pathway.

Small molecule antagonists of Tcf4/beta-catenin complex inhibit the growth of HCC cells in vitro and in vivo.

Hepatocellular carcinoma (HCC) is the 5th most common cancer worldwide. It is intrinsically resistant toward standard chemotherapy, making it imperative to develop novel selective chemotherapeutic agents. The Wnt/beta-catenin pathway plays critical roles in development and oncogenesis, and is dysregulated in HCC. Our study aims to evaluate the activity of 3 small molecule antagonists of the Tcf4/beta-catenin complex (PKF118-310, PKF115-584 and CGP049090) on HCC cell lines in vitro and in vivo. All 3 chemicals displayed dose-dependent cytotoxicity in vitro against all 3 HCC cell lines (HepG2, Hep40 and Huh7), but were at least 10 times less cytotoxic to normal hepatocytes (from 3 donors) by using ATP assay. In HepG2 and Huh7 cells, treatment with the antagonists decreased Tcf4/beta-catenin binding capability and transcriptional activity, associated with downregulation of the endogenous Tcf4/ beta-catenin target genes c-Myc, cyclin D1 and survivin. In HepG2 and Huh7 cells, treatment with the antagonists induced apoptosis and cell cycle arrest at the G1/S phase. All antagonists suppressed in vivo tumor growth in a HepG2 xenograft model, associated with apoptosis and reduced c-Myc, cyclin D1 and survivin expressions. Our results suggest that these 3 antagonists of the Tcf4/beta-catenin complex are potential chemotherapeutic agents which may offer a pathway specific option for the clinical management of HCC.

Design and synthesis of novel diaminoquinazolines with in vivo efficacy for beta-catenin/T-cell transcriptional factor 4 pathway inhibition.

We are introducing a novel series of 2,4-diaminoquinazolines as beta-catenin/Tcf4 inhibitors which were identified by ligand-based design. Here we elucidate the SAR of this series and explain how we were able to improve key molecular properties such as solubility and cLogP leading to compound 9. Analogue 9 exhibited better biological activity and improved physical and pharmacological properties relative to the HTS hit 49. Furthermore, 9 demonstrated good cell growth inhibition against several human colorectal cancer lines such as LoVo and HT29. In addition, treatment with compound 9 led to gene expression changes that overlapped significantly with the transcriptional profile resulting from the pathway inhibition by siRNA knockdown of beta-catenin or Tcf4. Subsequently, 9 was tested for efficacy in a beta-catenin/RKE-mouse xenograft, where it led to more then 50% decrease in tumor volume.

Stabilizers of the Max homodimer identified in virtual ligand screening inhibit Myc function.

Many human cancers show constitutive or amplified expression of the transcriptional regulator and oncoprotein Myc, making Myc a potential target for therapeutic intervention. Here we report the down-regulation of Myc activity by reducing the availability of Max, the essential dimerization partner of Myc. Max is expressed constitutively and can form unstable homodimers. We have isolated stabilizers of the Max homodimer by applying virtual ligand screening (VLS) to identify specific binding pockets for small molecule interactors. Candidate compounds found by VLS were screened by fluorescence resonance energy transfer, and from these screens emerged a potent, specific stabilizer of the Max homodimer. In vitro binding assays demonstrated that the stabilizer enhances the formation of the Max-Max homodimer and interferes with the heterodimerization of Myc and Max in a dose-dependent manner. Furthermore, this compound interferes with Myc-induced oncogenic transformation, Myc-dependent cell growth, and Myc-mediated transcriptional activation. The Max-Max stabilizer can be considered a lead compound for the development of inhibitors of the Myc network.