Linda Z. Penn

Induction of apoptosis in fibroblasts by c-myc protein

Although Rat-1 fibroblasts expressing c-myc constitutively are unable to arrest growth in low serum, their numbers do not increase in culture because of substantial cell death. We show this cell death to be dependent upon expression of c-myc protein and to occur by apoptosis. Regions of the c-myc protein required for induction of apoptosis overlap with regions necessary for cotransformation, autoregulation, and inhibition of differentiation, suggesting that the apoptotic function of c-myc protein is related to its other functions. Moreover, cells with higher levels of c-myc protein are more prone to cell death upon serum deprivation. Finally, we demonstrate that deregulated c-myc expression induces apoptosis in cells growth arrested by a variety of means and at various points in the cell cycle.

Reflecting on 25 years with MYC

Just over 25 years ago, MYC, the human homologue of a retroviral oncogene, was identified. Since that time, MYC research has been intense and the advances impressive. On reflection, it is astonishing how each incremental insight into MYC regulation and function has also had an impact on numerous biological disciplines, including our understanding of molecular oncogenesis in general. Here we chronicle the major advances in our understanding of MYC biology, and peer into the future of MYC research.

Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis

Bax promotes cell death by permeabilizing mitochondrial outer membranes by an unresolved mechanism. However, in cells lacking the gene c‐myc, membrane permeabilization by Bax is blocked by changes in the mitochondria that prevent Bax oligomerization. Drug‐treated c‐myc null cells and cells expressing Myc were used to map the topology of Bax in membranes prior to and after mitochondrial permeabilization. Chemical labeling of single cysteine mutants of Bax using a membrane bilayer impermeant cysteine‐specific modifying agent revealed that Bax inserted both the ‘pore domain’ (helices α5–α6), and the tail‐anchor (helix α9) into membranes prior to oligomerization and membrane permeabilization. Additional topology changes for Bax were not required in Myc‐expressing cells to promote oligomerization and cytochrome c release. Our results suggest that unlike most pore‐forming proteins, Bax membrane permeabilization results from oligomerization of transmembrane monomers rather than concerted insertion of the pore domains of a preformed oligomer.

Negative autoregulation of c-myc transcription.

The introduction of activated c‐myc and v‐myc genes into a variety of non‐established and established cells results in the suppression of endogenous c‐myc expression. As measured in Rat‐1 fibroblasts, the suppression occurs at the level of transcriptional initiation. Moreover, the extent of the down‐regulation is proportional to the cellular concentration of c‐myc protein, and the critical concentration range in which the endogenous c‐myc RNA is effectively suppressed corresponds to that found in non‐transformed cells. In addition, the autoregulatory mechanism is not only dependent on c‐myc protein, but also requires additional trans‐acting factors. These results support a role for c‐myc in the regulation of cellular gene transcription and suggest that a negative feedback mechanism can act as a homeostatic regulator of c‐myc expression in vivo.

Bcl-xL/Bcl-2 coordinately regulates apoptosis, cell cycle arrest and cell cycle entry.

Bcl‐xL and Bcl‐2 inhibit both apoptosis and proliferation. In investigating the relationship between these two functions of Bcl‐xL and Bcl‐2, an analysis of 24 Bcl‐xL and Bcl‐2 mutant alleles, including substitutions at residue Y28 previously reported to selectively abolish the cell cycle activity, showed that cell cycle delay and anti‐apoptosis co‐segregated in all cases. In determining whether Bcl‐2 and Bcl‐xL act in G0 or G1, forward scatter and pyronin Y fluorescence measurements indicated that Bcl‐2 and Bcl‐xL cells arrested more effectively in G0 than controls, and were delayed in G0–G1 transition. The cell cycle effects of Bcl‐2 and Bcl‐xL were reversed by Bad, a molecule that counters the survival function of Bcl‐2 and Bcl‐xL. When control and Bcl‐xL cells of equivalent size and pyronin Y fluorescence were compared, the kinetics of cell cycle entry were similar, demonstrating that the ability of Bcl‐xL and Bcl‐2 cells to enhance G0 arrest contributes significantly to cell cycle delay. Our data suggest that cell cycle effects and increased survival both result from intrinsic functions of Bcl‐2 and Bcl‐xL.

Identifying Genes Regulated in a Myc-dependent Manner

The c-myc proto-oncogene can direct a diverse array of biological activities, including cell cycle progression, apoptosis, and differentiation. It is believed that Myc can affect this wide variety of activities by functioning as a regulator of gene transcription, although few targets have been identified to date. To delineate the molecular program regulated downstream of Myc, we used a cDNA microarray approach and identified 52 putative targets out of >6000 cDNAs analyzed. To further distinguish the subset of genes whose regulation was dependent upon Myc per se from those regulated in response to activation of general mitogenic or apoptotic programs, the putative cDNA targets were then screened by a series of assays. By this approach 37 putative targets were ruled out and 15 Myc target genes were uncovered. Interestingly, comparing our results with other high throughput screens reveals that certain putative Myc targets previously reported are shown not to be regulated downstream of Myc (e.g. ribosomal proteins, HSP90β), whereas others are further supported by our analyses (e.g. pdgfβr, nucleolin). The identity of genes specifically regulated downstream of Myc provides the critical tools required to understand the role Myc holds in the transformation process and to delineate how Myc functions as a regulator of gene transcription.

CUL7 Is a Novel Antiapoptotic Oncogene

Using an expression cloning approach, we identify CUL7, a member of the cullin family, as a functional inhibitor of Myc-induced apoptosis. Deregulated expression of the Myc oncogene drives cellular proliferation yet also sensitizes cells to undergo p53-dependent and p53-independent apoptosis. Here, we report that CUL7 exerts its antiapoptotic function through p53. CUL7 binds directly to p53, and small interfering RNA-mediated knockdown of CUL7 results in the elevation of p53 protein levels. This antiapoptotic role of CUL7 enables this novel oncogene to cooperate with Myc to drive transformation. Deregulated ectopic expression of c-Myc and CUL7 promotes Rat1a cell growth in soft agar, and knockdown of CUL7 significantly blocks human neuroblastoma SHEP cell growth in an anchorage-independent manner. Furthermore, using public microarray data sets, we show that CUL7 mRNA is significantly overexpressed in non-small cell lung carcinoma and is associated with poor patient prognosis. We provide experimental evidence to show CUL7 is a new oncogene that cooperates with Myc in transformation by blocking Myc-induced apoptosis in a p53-dependent manner.

Exploiting the mevalonate pathway to distinguish statin-sensitive multiple myeloma

Statin inhibitors, used to control hypercholesterolemia, trigger apoptosis of hematologic tumor cells and therefore have immediate potential as anticancer agents. Evaluations of statins in acute myelogenous leukemia and multiple myeloma have shown that statin efficacy is mixed, with only a subset of tumor cells being highly responsive. Our goal was to distinguish molecular features of statin-sensitive and -insensitive myeloma cells and gain insight into potential predictive markers. We show that dysregulation of the mevalonate pathway is a key determinant of sensitivity to statin-induced apoptosis in multiple myeloma. In sensitive cells, the classic feedback response to statin exposure is lost. This results in deficient up-regulation of 2 isoforms of hydroxymethylglutaryl coenzyme A reductase: the rate-limiting enzyme of the mevalonate pathway and hydroxymethylglutaryl coenzyme A synthase 1. To ascertain the clinical utility of these findings, we demonstrate that a subset of primary myeloma cells is sensitive to statins and that monitoring dysregulation of the mevalonate pathway may distinguish these cancers. We also show statins are highly effective and well tolerated in an orthotopic model of myeloma using cells harboring this dysregulation. This determinant of sensitivity further provides molecular rationale for the significant therapeutic index of statins on these tumor cells.

Determinants of sensitivity to lovastatin-induced apoptosis in multiple myeloma

Statins, commonly used to treat hypercholesterolemia, have been shown to trigger tumor-specific apoptosis in certain cancers, including multiple myeloma (MM), a plasma cell malignancy with poor prognosis. In this article, we show that of a panel of 17 genetically distinct MM cell lines, half were sensitive to statin-induced apoptosis and, despite pharmacodynamic evidence of drug uptake and activity, the remainder were insensitive. Sensitive cells were rescued from lovastatin-induced apoptosis by mevalonate, geranylgeranyl PPi, and partially by farnesyl PPi, highlighting the importance of isoprenylation. Expression profiling revealed that Rho GTPase mRNAs were differentially expressed upon lovastatin exposure in sensitive cells, yet ectopic expression of constitutively active Rho or Ras proteins was insufficient to alter sensitivity to lovastatin-induced apoptosis. This suggests that sensitivity involves more than one isoprenylated protein and that statins trigger apoptosis by blocking many signaling cascades, directly or indirectly deregulated by the oncogenic lesions of the tumor cell. Indeed, clustering on the basis of genetic abnormalities was shown to be significantly associated with sensitivity (P = 0.003). These results suggest that statins may be a useful molecular targeted therapy in the treatment of a subset of MM. [Mol Cancer Ther 2007;6(6):1886–97]

Myc The Beauty and the Beast

The iconic history of the Myc oncoprotein encompasses 3 decades of intense scientific discovery. There is no question that Myc has been a pioneer, advancing insight into the molecular basis of cancer as well as functioning as a critical control center for several diverse biological processes and regulatory mechanisms. This narrative chronicles the journey and milestones that have defined the understanding of Myc, and it provides an opportunity to consider future directions in this challenging yet rewarding field.