Myc overexpression brings out unexpected antiapoptotic effects of miR-34a.

Downregulation of microRNA-34a by Myc is known to be essential for tumorigenesis and improve tumor-cell survival. Conversely, upregulation of miR-34a by p53 is thought to enhance its acetylation and activity and contribute to the pro-apoptotic effects of this tumor suppressor. We sought to determine whether restoration of miR-34a levels in B-lymphoid cells with Myc overexpression would aid therapeutic apoptosis. Unexpectedly, delivery of miR-34a, which doesn't target p53 directly, severely compromised steady-state p53 levels. This effect was preceded and mediated by direct targeting of Myc, which sustained p53 protein levels via the Arf-Hdm2 pathway. As a result, in the presence of Myc, miR-34a inhibited p53-dependent bortezomib-induced apoptosis as efficiently as anti-p53 small interfering RNA. Conversely, inhibition of miR-34a using antisense RNA sensitized lymphoma cells to therapeutic apoptosis. Thus, in tumors with deregulated Myc expression, miR-34a confers drug resistance and could be considered a therapeutic target.

Oncogenic BRAF regulates beta-Trcp expression and NF-kappaB activity in human melanoma cells.

Mutational activation of BRAF is a frequent event in human malignant melanomas suggesting that BRAF-dependent signaling is conducive to melanoma cell growth and survival. Previously published work reported that melanoma cells exhibit constitutive anti-apoptotic nuclear factor kappaB (NF-kappaB) transcription factor activation triggered by proteolysis of its inhibitor IkappaB. IkappaB degradation is dependent upon its phosphorylation by the IkappaB kinase (IKK) complex and subsequent ubiquitination facilitated by beta-Trcp E3 ubiquitin ligase. Here, we report that melanocytes expressing a conditionally oncogenic form of BRAF(V600E) exhibit enhanced beta-Trcp expression, increased IKK activity and a concomitant increase in the rate of IkappaBalpha degradation. Conversely, inhibition of BRAF signaling using either a broad-spectrum Raf inhibitor (BAY 43-9006) or by selective knock-down of BRAF(V600E) expression by RNA interference in human melanoma cells leads to decreased IKK activity and beta-Trcp expression, stabilization of IkappaB, inhibition of NF-kappaB transcriptional activity and sensitization of these cells to apoptosis. Taken together, these data support a model in which mutational activation of BRAF in human melanomas contributes to constitutive induction of NF-kappaB activity and to increased survival of melanoma cells.

Intratumoral delivery of an interferon gamma retrovirus-producing cells inhibits growth of a murine melanoma by a non-immune mechanism.

Interferon gamma (IFNgamma) is a potent inhibitor of cell growth effective against a wide range of tumor-derived cell lines. We cloned murine IFNgamma cDNA into a retroviral vector and created a packaging cell line (Am-gamma) producing this IFNgamma-encoding retrovirus. In a pilot experiment, admixing and co-injection of equal numbers of retrovirus-producing and target B16 melanoma cells led to high rates of infection and strong suppression of neoplastic growth. This effect was observed in the absence of measurable systemic production of IFNgamma and could be reproduced in animals lacking cytotoxic immune responses. Tumor angiogenesis was unaffected and no increase in apoptosis was apparent; however, mitotic indices were greatly reduced in Am-gamma-containing abortive tumors. We thus concluded that IFNgamma directly affects proliferation of B16 cells. Indeed, exposure of B16 cells to IFNgamma in vitro inhibits cell division, as measured by a thymidine incorporation assay. Most importantly, repeated intratumoral injections of Am-gamma stunted growth of established B16 melanomas in 75% of treated animals. Thus, this approach can serve as a prototype for new anti-cancer modalities.

Cutting edge: systemic inhibition of angiogenesis underlies resistance to tumors during acute toxoplasmosis.

The ability of various infections to suppress neoplastic growth has been well documented. This phenomenon has been traditionally attributed to infection-induced concomitant, cell-mediated antitumor immunity. We found that infection with Toxoplasma gondii effectively blocked neoplastic growth of a nonimmunogenic B16.F10 melanoma. Moreover, this effect was independent of cytotoxic T or NK cells, production of NO by macrophages, or the function of the cytokines IL-12 and TNF-alpha. These findings suggested that antitumor cytotoxicity was not the primary mechanism of resistance. However, infection was accompanied by strong, systemic suppression of angiogenesis, both in a model system and inside the nascent tumor. This suppression resulted in severe hypoxia and avascular necrosis that are incompatible with progressive neoplastic growth. Our results identify the suppression of tumor neovascularization as a novel mechanism critical for infection-induced resistance to tumors.

Activation of the myc oncoprotein leads to increased turnover of thrombospondin-1 mRNA.

The Myc oncoprotein is implicated in transcriptional regulation of a variety of genes pertaining to cell cycle and neoplastic transformation. Examples of both positive and negative regulation have been reported that involve E-box and initiator (Inr) promoter elements, respectively. In both cases, Myc is thought to induce changes in transcription initiation. We have previously shown that overexpression of Myc causes down-regulation of the thrombospondin-1 (tsp-1) gene, an important negative modulator of tumor angiogenesis. In this study, we demonstrate that Myc in combination with Max can bind, albeit with low affinity, to an E-box-like element in the tsp-1 promoter. However, the 2.7 kb DNA segment containing both this non-canonical E-box and an Inr-like sequence does not constitute a Myc-responsive element in a transient expression system. Furthermore, Myc does not significantly affect the rate of initiation or elongation of the tsp-1 mRNA. Thus, in this instance Myc does not act as a canonical transcription factor. Instead, as demonstrated by blocking de novo RNA synthesis, down-regulation of the tsp-1 gene by Myc occurs through increased mRNA turnover. To our knowledge, this is the first example of gene regulation by Myc that involves mRNA destabilization.

An in vivo function for the transforming Myc protein: elicitation of the angiogenic phenotype.

The ability of neoplastic cells to recruit blood vasculature is crucial to their survival in the host organism. However, the evidence linking dominant oncogenes to the angiogenic switch remains incomplete. We demonstrate here that Myc, an oncoprotein implicated in many human malignancies, stimulates neovascularization. As an experimental model, we used Rat-1A fibroblasts that form vascular tumors upon transformation by Myc in immunocompromised mice. Our previous work and the use of neutralizing antibodies reveal that in these cells, the angiogenic switch is achieved via down-modulation of thrombospondin-1, a secreted inhibitor of angiogenesis, whereas the levels of vascular endothelial growth factor, a major activator of angiogenesis, remain high and unaffected by Myc. Consistent with this finding, overexpression of Myc confers upon the conditioned media the ability to promote migration of adjacent endothelial cells in vitro and corneal neovascularization in vivo. Furthermore, mobilization of estrogen-dependent Myc in vivo with the appropriate steroid provokes neovascularization of cell implants embedded in Matrigel. These data suggest that Myc is fully competent to trigger the angiogenic switch in vivo and that secondary events may not be required for neovascularization of Myc-induced tumors.