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Colorectal cancer (CRC) severely threatens human health and life span. An effective therapeutic strategy has not been established because we do not clearly know its pathogenesis. Here, we report that ceramide and sterol O-acyltransferase 1 (SOAT1) have roles in both spontaneous and chemical-induced intestinal cancers. We first found that miRNA-148a deficiency dramatically increased mouse gut dysbiosis through upregulating ceramide synthase 5 (Cers5) expression, which promoted ceramide synthesis afterward. The newly generated ceramide further promoted both azoxymethane/dextran sodium sulfate-induced (AOM/DSS-induced) and ApcMin/+ spontaneous intestinal tumorigenesis via increasing mouse gut dysbiosis. Meanwhile, increased level of ceramide correlated with the significant enhancements of both β-catenin activity and colorectal tumorigenesis in a TLR4-dependent fashion. Next, we found a direct binding of β-catenin to SOAT1 promoter to activate transcriptional expression of SOAT1, which further induced cholesterol esterification and colorectal tumorigenesis. In human patients with CRC, the same CERS5/TLR4/β-catenin/SOAT1 axis was also found to be dysregulated. Finally, the SOAT1 inhibitor (avasimibe) showed significant levels of therapeutic effects on both AOM/DSS-induced and ApcMin/+ spontaneous intestinal cancer. Our study clarified that ceramide promoted CRC development through increasing gut dysbiosis, further resulting in the increase of cholesterol esterification in a SOAT1-dependent way. Treatment with avasimibe to specifically decrease cholesterol esterification could be considered as a clinical strategy for effective CRC therapy in a future study.

Deregulation of c-Myc (Myc) occurs in many cancers. In addition to transforming various cell types, Myc also influences additional transformation-associated cellular phenotypes including proliferation, survival, genomic instability, reactive oxygen species production, and metabolism. Although Myc is wild type in most cancers (wtMyc), it occasionally acquires point mutations in certain lymphomas. Some of these mutations confer a survival advantage despite partially attenuating proliferation and transformation. Here, we have evaluated four naturally-occurring or synthetic point mutations of Myc for their ability to affect these phenotypes, as well as to promote genomic instability, to generate reactive oxygen species and to up-regulate aerobic glycolysis and oxidative phosphorylation. Our findings indicate that many of these phenotypes are genetically and functionally independent of one another and are not necessary for transformation. Specifically, the higher rate of glucose metabolism known to be associated with wtMyc deregulation was found to be independent of transformation. One mutation (Q131R) was greatly impaired for nearly all of the studied Myc phenotypes, yet was able to retain some ability to transform. These findings indicate that, while the Myc phenotypes examined here make additive contributions to transformation, none, with the possible exception of increased reliance on extracellular glutamine for survival, are necessary for achieving this state.

Glycoprotein Ib alpha (GpIbalpha), a trans-membrane glycoprotein, is expressed on the surface of megakaryocytes and platelets, where, in association with glycoprotein Ib beta, glycoprotein V, and glycoprotein IX, it normally forms the von Willebrand factor receptor (vWFR). A fully functional vWFR is necessary for platelet attachment, aggregation, and activation and has also been shown to regulate megakaryocyte ploidy. We have recently shown that the gene encoding GpIbalpha is a transcriptional target for the c-Myc oncoprotein and is more widely expressed than previously thought, with particularly high levels occurring in transformed cells. Indeed, GpIbalpha can substitute for c-Myc in promoting growth, transformation, and genomic instability. In the current work, we have demonstrated that, despite the promiscuous expression of GpIbalpha, other vWFR subunits remain largely restricted to megakaryocytes. We have characterized a panel of GpIbalpha mutants and shown that some regions of the protein essential for vWFR activity are not necessary for c-Myc-like functions. Specifically, the six C-terminal amino acids of the cytoplasmic domain, which mediate vWFR signaling, are entirely dispensible for the c-Myc-like functions of GpIbalpha. Instead, these require a more membrane-proximal filamin-binding domain. Also important is the GpIbalpha signal peptide, which, in the absence of other vWFR subunits, directs GpIbalpha to the endoplasmic reticulum rather than the membrane. Together, these results provide strong evidence that the domains of GpIbalpha mediating c-Myc-like functions are modular, genetically distinct, and independent of those involved in vWFR signaling.

CMYC has long been known to be among the most frequently de-regulated oncogenes in human cancer. Only recently, however has a clear understanding begun to emerge of how it promotes transformation. Through its role as a transcription factor, c-Myc alters the expression of hundreds of target genes, many of which are themselves oncogenes or tumor suppressors. The deregulation of c-Myc is both necessary and sufficient for the "acute" type of rapid in vitro transformation that occurs in certain established rodent cell lines. Transformation of primary rodent cells in vitro is also rapid but requires the contribution of at least one additional cooperating oncogene such as Ras. In contrast, the "chronic" form of in vivo transformation by c-Myc is a rare event that requires the acquisition of multiple mutations in other genes affecting cell cycle, senescence, and apoptosis. By greatly accelerating the intrinsic mutation rate at several levels, c-Myc increases the likelihood that these additional mutational "hits" will occur. Among the types of genomic instability mediated by c-Myc are single nucleotide substitutions and double-stranded breaks arising via the induction of reactive oxygen species, gene amplification and the generation of extrachromosomal elements, and numerical chromosomal defects resulting from aberrant DNA synthesis and defects in the mitotic spindle checkpoint. These non-mutually exclusive activities ensure a constant and varied source of genotoxic insults and suggest that c-Myc over-expression imposes a "mutator phenotype". This may be an early and necessary requirement for the initial steps in chronic transformation as well as for subsequent evolutionary changes that produce important tumor behaviors such as invasiveness, metastasis, and acquisition of chemotherapy resistance.

GpIbalpha, a subunit of the von Willebrand factor receptor, functions during blood clotting to promote platelet adhesion and activation. GpIbalpha is widely expressed, is positively regulated by c-Myc and is essential for the promotion of c-Myc-mediated chromosomal instability. We now show that GpIbalpha is also a classical oncoprotein in which its deregulated expression leads to transformation, reduced growth factor requirements, increased resistance to apoptosis, and, in primary cells, p53-dependent senescence. Finally, GpIbalpha also promotes double-stranded DNA breaks, and induces profound nuclear dysmorphology, indicating that, in addition to its direct transforming function, it displays genotoxicity at several distinct levels. These findings identify novel functions for GpIbalpha and pathways through which c-Myc mediates transformation and global genomic destabilization.

Members of the HMGA1 (high mobility group A1) family of architectural transcription factors, HMGA1a and HMGA1b, play important roles in many normal cellular processes and in tumorigenesis. We performed a yeast two-hybrid screen for HMGA1-interacting proteins and identified the SUMO E2 conjugase Ubc9 as one such partner. The Ubc9-interacting domain of HMGA1 is bipartite, consisting of a proline-rich region near the N terminus and an acidic domain at the extreme C terminus, whereas the HMGA1-interacting domain of Ubc9 comprises a single region previously shown to associate with and SUMOylate other transcription factors. Consistent with these findings, endogenous HMGA1 proteins and Ubc9 could be co-immunoprecipitated from several human cell lines. Studies with HMGA1b proteins containing mutations of either or both Ubc9-interacting domains and with Ubc9-depleted cell lines indicated that the proline-rich domain of HMGA1b positively influences transformation and growth, whereas the acidic domain negatively influences these properties. None of the changes in HMGA1 protein functions mediated by Ubc9 appears to require SUMOylation. These findings are consistent with the idea that Ubc9 can act as both a positive and negative regulator of proliferation and transformation via its non-SUMO-dependent interaction with HMGA1 proteins.

We recently reported (1991, Mol. Cell Biol. 11, 3699-3710) that depletion of c-myc protein by myc antisense sequences in ras-transformed NIH-3T3 cells reverses several of the malignant characteristics of these cells. These include transformed morphology, growth in soft agar, and ability to form tumors in athymic mice. In the present study we examined the same cells for in vitro adhesive behavior. Cells depleted of c-myc protein by antisense transfection showed no change in attachment to fibronectin-coated dishes as compared to ras-transformed NIH-3T3 cells but had greatly increased resistance to trypsin/EDTA-mediated release from the substratum after attachment. In concomitant studies, the cells were examined for fibronectin biosynthesis and cell surface fibronectin. There was no overall change in fibronectin biosynthesis in the c-myc antisense transfected cells as compared to the ras-transformed NIH-3T3. However, immunofluorescence staining revealed increased amount of surface fibronectin associated with the antisense c-myc-expressing transfectants. Taken together, these data indicate that the conditional reacquisition of the nonmalignant phenotype in ras-transformed NIH-3T3 cells by selected depletion of c-myc protein is associated with an increase in cell-substrate adhesion. This, in turn, is associated with an increase in surface fibronectin.

ras oncogene-transformed NIH 3T3 cells expressing glucocorticoid-inducible antisense c-myc cDNA transcripts at levels sufficient to deplete c-myc protein lost their transformed morphology and the ability to grow in soft agar; their ability to form tumors in nude mice was also impaired. These changes were dependent on the continuous expression of the antisense sequences. No major effects on plating efficiencies, growth rates in monolayer culture, or immortalization were observed in the revertant cells, indicating that the observed effects were not a toxic consequence of c-myc protein depletion. Transfection with the same vector expressing c-myc in the sense orientation or other control vectors had no effect on transformation. These results suggest that a certain minimum level of expression of c-myc is required for the maintenance of ras transformation in NIH 3T3 cells.

Several different proto-oncogenes have been shown to influence cellular differentiation. One of the most widely studied model systems has been the Friend murine erythroleukemia cell (F-MELC) line, which can be induced to undergo erythroid differentiation by a variety of chemical agents. Constitutive overexpression of either the c-myc or c-myb proto-oncogenes has been previously shown to inhibit F-MELC differentiation, whereas c-myc antisense sequences accelerate the process. To investigate the potential involvement of other proto-oncogenes and immediate early response genes in F-MELC differentiation, we studied the expression of the three known members of the jun family as well as another gene, egr-1, which, like the jun family members, is expressed as an immediate early response gene in growth factor-stimulated quiescent cells. All four genes were expressed in F-MELC, although the levels of expression and modes of regulation differed. Transfection with amplifiable c-jun, junB, or junD expression plasmids inhibited differentiation, whereas transfection with an egr-1 expression plasmid was without effect. These results indicate that jun family members play a role in mediating F-MELC differentiation. The known inhibitory effect of phorbol ester tumor promoters on F-MELC differentiation may be the result of their known stimulation of jun expression.