Analogous to the c-Myc (Myc)/Max family of bHLH-ZIP transcription factors, there exists a parallel regulatory network of structurally and functionally related proteins with Myc-like functions. Two related Myc-like paralogs, termed MondoA and MondoB/carbohydrate response element-binding protein (ChREBP), up-regulate gene expression in heterodimeric association with the bHLH-ZIP Max-like factor Mlx. Myc is necessary to support liver cancer growth, but not for normal hepatocyte proliferation. Here, we investigated ChREBP's role in these processes and its relationship to Myc. Unlike Myc loss, ChREBP loss conferred a proliferative disadvantage to normal murine hepatocytes, as did the combined loss of ChREBP and Myc. Moreover, hepatoblastomas (HBs) originating in ,, or / backgrounds grew significantly more slowly. Metabolic studies on livers and HBs in all three genetic backgrounds revealed marked differences in oxidative phosphorylation, fatty acid β-oxidation (FAO), and pyruvate dehydrogenase activity. RNA-Seq of livers and HBs suggested seven distinct mechanisms of Myc-ChREBP target gene regulation. Gene ontology analysis indicated that many transcripts deregulated in the background encode enzymes functioning in glycolysis, the TCA cycle, and β- and ω-FAO, whereas those dysregulated in the background encode enzymes functioning in glycolysis, glutaminolysis, and sterol biosynthesis. In the / background, additional deregulated transcripts included those involved in peroxisomal β- and α-FAO. Finally, we observed that Myc and ChREBP cooperatively up-regulated virtually all ribosomal protein genes. Our findings define the individual and cooperative proliferative, metabolic, and transcriptional roles for the "Extended Myc Network" under both normal and neoplastic conditions.

Edited by Eric FearonHepatoblastoma (HB) is associated with aberrant activation of the ␤-catenin and Hippo/YAP signaling pathways. Overexpression of mutant ␤-catenin and YAP in mice induces HBs that express high levels of c-Myc (Myc). In light of recent observations that Myc is unnecessary for long-term hepatocyte proliferation, we have now examined its role in HB pathogenesis using the above model. Although Myc was found to be dispensable for in vivo HB initiation, it was necessary to sustain rapid tumor growth. Gene expression profiling identified key molecular differences between myc ؉/؉ (WT) and myc ؊/؊ (KO) hepatocytes and HBs that explain these behaviors. In HBs, these included both Myc-dependent and Myc-independent increases in families of transcripts encoding ribosomal proteins, non-structural factors affecting ribosome assembly and function, and enzymes catalyzing glycolysis and lipid bio-synthesis. In contrast, transcripts encoding enzymes involved in fatty acid ␤-oxidation were mostly down-regulated. Myc-independent metabolic changes associated with HBs included dramatic reductions in mitochondrial mass and oxidative function, increases in ATP content and pyruvate dehydrogenase activity, and marked inhibition of fatty acid ␤-oxidation (FAO). Myc-dependent metabolic changes included higher levels of neutral lipid and acetylCoA in WT tumors. The latter correlated with higher histone H3 acetylation. Collectively, our results indicate that the role of Myc in HB pathogenesis is to impose mutually dependent changes in gene expression and metabolic reprogramming that are unattainable in non-transformed cells and that cooperate to maximize tumor growth.

Hepatoblastoma (HB) is the most common pediatric liver cancer. Although long-term survival of HB is generally favorable, it depends on clinical stage, tumor histology, and a variety of biochemical and molecular features. HB appears almost exclusively before the age of 3 years, is represented by seven histological subtypes, and is usually associated with highly heterogeneous somatic mutations in the catenin β1 (CTNNB1) gene, which encodes β-catenin, a Wnt ligand–responsive transcriptional co-factor. Numerous recurring β-catenin mutations, not previously documented in HB, have also been identified in various other pediatric and adult cancer types. Little is known about the underlying factors that determine the above HB features and behaviors or whether non-HB–associated β-catenin mutations are tumorigenic when expressed in hepatocytes. Here, we investigated the oncogenic properties of 14 different HB– and non-HB–associated β-catenin mutants encoded by Sleeping Beauty vectors following their delivery into the mouse liver by hydrodynamic tail-vein injection. We show that all β-catenin mutations, as well as WT β-catenin, are tumorigenic when co-expressed with a mutant form of yes-associated protein (YAP). However, tumor growth rates, histologies, nuclear-to-cytoplasmic partitioning, and metabolic and transcriptional landscapes were strongly influenced by the identities of the β-catenin mutations. These findings provide a context for understanding at the molecular level the notable biological diversity of HB.

Well-differentiated liposarcoma/atypical lipomatous tumor and dedifferentiated liposarcoma can be difficult to distinguish from benign lipomatous neoplasms and other high-grade sarcomas, respectively. Cytogenetics in these tumors has identified ring and giant chromosomes composed of 12q13-15 amplicons including the MDM2 gene. Identifying MDM2 amplification by fluorescence in situ hybridization may prove an adjunctive tool in the diagnosis of lipomatous neoplasms. Dual color fluorescence in situ hybridization employing a laboratorydeveloped BAC label probe cocktail specific for MDM2 (12q15) and a probe for the centromeric region of chromosome 12 (Abbott Molecular, DesPlaines, IL) was performed on formalin-fixed and paraffin-embedded tissue including whole sections from atypical lipomatous tumors (n ¼ 13), dedifferentiated liposarcomas (n ¼ 14), benign lipomatous tumors (n ¼ 30), and pleomorphic sarcoma, not otherwise specified (n ¼ 10), and a tissue microarray containing a variety of high-grade sarcomas (n ¼ 63). An MDM2/chromosome 12 ratio Z2.0 was considered amplified, o2.0 nonamplified, and cases displaying 42 signals of both probes and an MDM2 ratio o2.0 polysomic for chromosome 12. Of the well-differentiated and dedifferentiated liposarcomas, 100% showed amplification of MDM2. Chromosome 12 polysomy was noted in 89% of spindle cell/pleomorphic lipomas, while all angiolipomas and lipomas were nonamplified and eusomic. MDM2 amplification was observed in 40% of pleomorphic sarcomas and a small subset of high-grade sarcomas (3/63). MDM2/chromosome 12 fluorescence in situ hybridization is a sensitive and specific tool (both 100%) in evaluating low-grade lipomatous neoplasms. The specificity decreases in high-grade sarcomas, as MDM2 amplification was observed in a small portion of pleomorphic sarcomas and high-grade sarcomas other than dedifferentiated liposarcomas. Importantly, none of the benign lipomatous lesions were MDM2 amplified and even cells in areas of well-differentiated liposarcomas with minimal cytologic atypia were amplified, making the probe a valuable tool in the diagnosis of even limited biopsy samples of well-differentiated lipomatous neoplasms.

Hepatocellular carcinoma (HCC) is a common cancer that frequently overexpresses the c-Myc (Myc) oncoprotein. Using a mouse model of Myc-induced HCC, we studied the metabolic, biochemical, and molecular changes accompanying HCC progression, regression, and recurrence. These involved altered rates of pyruvate and fatty acid β-oxidation and the likely re-directing of glutamine into biosynthetic rather than energy-generating pathways. Initial tumors also showed reduced mitochondrial mass and differential contributions of electron transport chain complexes I and II to respiration. The uncoupling of complex II's electron transport function from its succinate dehydrogenase activity also suggested a mechanism by which Myc generates reactive oxygen species. RNA sequence studies revealed an orderly progression of transcriptional changes involving pathways pertinent to DNA damage repair, cell cycle progression, insulin-like growth factor signaling, innate immunity, and further metabolic re-programming. Only a subset of functions deregulated in initial tumors was similarly deregulated in recurrent tumors thereby indicating that the latter can "normalize" some behaviors to suit their needs. An interactive and freely available software tool was developed to allow continued analyses of these and other transcriptional profiles. Collectively, these studies define the metabolic, biochemical, and molecular events accompanyingHCCevolution, regression, and recurrence in the absence of any potentially confounding therapies.

Rapidly proliferating cells increase glycolysis at the expense of oxidative phosphorylation (oxphos) to generate sufficient levels of glycolytic intermediates for use as anabolic substrates. The pyruvate dehydrogenase complex (PDC) is a critical mitochondrial enzyme that catalyzes pyruvate’s conversion to acetyl coenzyme A (AcCoA), thereby connecting these two pathways in response to complex energetic, enzymatic and metabolic cues. Here we utilized a mouse model of hepatocyte-specific PDC inactivation to determine the need for this metabolic link during normal hepatocyte regeneration and malignant transformation. In PDC “knockout” (KO) animals, the long-term regenerative potential of hepatocytes was unimpaired, and growth of aggressive experimental hepatoblastomas (HB) was only modestly slowed in the face of 80–90% reductions in AcCoA and significant alterations in the levels of key TCA cycle intermediates and amino acids. Overall, oxphos activity in KO livers and HB was comparable to that of control counterparts, with evidence that metabolic substrate abnormalities were compensated for by increased mitochondrial mass. These findings demonstrate that the biochemical link between glycolysis and the TCA cycle can be completely severed without affecting normal or neoplastic proliferation, even under the most demanding circumstances.

Interaction of integrins with the extracellular matrix leads to transmission of signals, cytoskeletal reorganizations, and changes in cell behavior. While many signaling molecules are known to be activated within Rac-induced focal complexes or Rho-induced focal adhesions, the way in which integrin-mediated adhesion leads to activation of Rac and Rho is not known. In the present study, we identified clusters of integrin that formed upstream of Rac activation. These clusters contained a Rac-binding protein(s) and appeared to be involved in Rac activation. The integrin clusters contained calpain and calpain-cleaved β3 integrin, while the focal complexes and focal adhesions that formed once Rac and Rho were activated did not. Moreover, the integrin clusters were dependent on calpain for their formation. In contrast, while Rac- and Rho-GTPases were dependent on calpain for their activation, formation of focal complexes and focal adhesions by constitutively active Rac or Rho, respectively, occurred even when calpain inhibitors were present. Taken together, these data are consistent with a model in which integrin-induced Rac activation requires the formation of integrin clusters. The clusters form in a calpain-dependent manner, contain calpain, calpain-cleaved integrin, and a Rac binding protein(s). Once Rac is activated, other integrin signaling complexes are formed by a calpain-independent mechanism(s).

Integrin-induced adhesion leads to cytoskeletal reorganizations, cell migration, spreading, proliferation, and differentiation. The details of the signaling events that induce these changes in cell behavior are not well understood but they appear to involve activation of Rho family members which activate signaling molecules such as tyrosine kinases, serine/threonine kinases, and lipid kinases. The result is the formation of focal complexes, focal adhesions, and bundles and networks of actin filaments that allow the cell to spread. The present study shows that -calpain is active in adherent cells, that it cleaves proteins known to be present in focal complexes and focal adhesions, and that overexpression of -calpain increased the cleavage of these proteins, induced an overspread morphology and induced an increased number of stress fibers and focal adhesions. Inhibition of calpain with membrane permeable inhibitors or by expression of a dominant negative form of -calpain resulted in an inability of cells to spread or to form focal adhesions, actin filament networks, or stress fibers. Cells expressing constitutively active Rac1 could still form focal complexes and actin filament networks (but not focal adhesions or stress fibers) in the presence of calpain inhibitors; cells expressing constitutively active RhoA could form focal adhesions and stress fibers. Taken together, these data indicate that calpain plays an important role in regulating the formation of focal adhesions and Rac-and Rho-induced cytoskeletal reorganizations and that it does so by acting at sites upstream of both Rac1 and RhoA.Adhesion of cells on integrin substrates results in cell spreading, migration, differentiation, and proliferation and is essential for events such as inflammation, platelet clot formation, development, and wound healing (1-3). Recent evidence has shown that one of the early events following integrin-ligand interactions is the clustering of integrins into small complexes with signaling molecules (4, 5). The subsequent activation of Cdc42 and Rac1 induces the polymerization of new actin filaments that organize into bundles and submembranous networks, thus, causing the extension of filopodia and lamellipodia (4 -8). The dynamic breakdown and formation of new focal complexes in the extending lamellipodia allows the cells to spread. RhoA then becomes activated and induces the formation of larger complexes of integrins, cytoskeletal proteins, and signaling molecules known as focal adhesions. RhoA also induces myosin to interact with actin filaments, causing the filaments to assemble into bundles known as stress fibers that terminate at the focal adhesions and allow tension to be generated on ligand-occupied integrin in the spreading cells (9 -12).By analogy to activation of Ras proteins by growth factor receptors, it is likely that Rho family members are activated by exchange factors that are recruited to sites of ligand-occupied integrin (13-19). However, little is known about the mechanisms inducing recruitment or activation of ...