Metastasis as a faulty recapitulation of ontogeny

RAF oncogenes are involved in a variety of phenotypic switch phenomena. If for example oncogenic RAF is expressed together with Myc in B lineage cells, a lineage switch to macrophages occurs at low frequency in vitro and in vivo. In addition, if RAF is expressed in type II alveolar epithelial cells slow growing lung adenomas are formed and a switch from columnar to cuboidal cells is detected in these mice upon p53 deletion. A similar switch is also seen, if ectopic Myc is present in our lung tumor mouse model. Moreover, in the liver of these mice with both oncogenes metastases are found. If E-cadherin function is impaired in our RAF-dependent lung tumor model, a switch from adenoma to adenocarcinoma occurs and genes characteristic for the early endodermal lineage are expressed. Based on these data I propose a novel model of metastasis and describe its implications. The hallmark of the model is the induction of a state of plasticity in tumor cells, which allows the reversal of differentiation to an earlier point in their ontogenic history

Absence of mutations in the coding sequence of the potential tumor suppressor 3pK in metastatic melanoma

BACKGROUND: Activation of Ras or Raf contributes to tumorigenesis of melanoma. However, constitutive Raf activation is also a characteristic of the majority of benign melanocytic nevi and high intensity signaling of either Ras or Raf was found to induce growth inhibition and senescence rather than transformation. Since the chromosome 3p kinase (3pK)) is a target of the Ras/Raf/Mek/Erk signaling pathway which antagonizes the function of the oncogene and anti-differentiation factor Bmi-1, 3pK may function as a tumor suppressor in tumors with constitutive Ras/Raf activation. Consequently, we tested whether inactivating 3pK mutations are present in melanoma. METHODS: 30 metastatic melanoma samples, which were positive for activating mutations of either BRaf or NRas, were analyzed for possible mutations in the 3pk gene. The 10 coding exons and their flanking intron sequences were amplified by PCR and direct sequencing of the PCR products was performed. RESULTS: This analysis revealed that besides the presence of some single nucleotide polymorphisms in the 3pk gene, we could not detect any possible loss of function mutation in any of these 30 metastatic melanoma samples selected for the presence of activating mutations within the Ras/Raf/Mek/Erk signaling pathway. CONCLUSION: Hence, in melanoma with constitutively active Ras/Raf inactivating mutations within the 3pk gene do not contribute to the oncogenic phenotype of this highly malignant tumor

Negative regulation of mitochondrial VDAC channels by C-Raf kinase

BACKGROUND: Growth of cancer cells results from the disturbance of positive and negative growth control mechanisms and the prolonged survival of these genetically altered cells due to the failure of cellular suicide programs. Genetic and biochemical approaches have identified Raf family serine/threonine kinases B-Raf and C-Raf as major mediators of cell survival. C-Raf cooperates with Bcl-2/Bcl-X(L) in suppression of apoptosis by a mechanism that involves targeting of C-Raf to the outer mitochondrial membrane and inactivation of the pro-apoptotic protein Bad. However, apoptosis suppression by C-Raf also occurs in cells lacking expression of Bad or Bcl-2. RESULTS: Here we show that even in the absence of Bcl-2/Bcl-X(L), mitochondria-targeted C-Raf inhibits cytochrome c release and caspase activation induced by growth factor withdrawal. To clarify the mechanism of Bcl-2 independent survival control by C-Raf at the mitochondria a search for novel mitochondrial targets was undertaken that identified voltage-dependent anion channel (VDAC), a mitochondrial protein (porin) involved in exchange of metabolites for oxidative phosphorylation. C-Raf forms a complex with VDAC in vivo and blocks reconstitution of VDAC channels in planar bilayer membranes in vitro. CONCLUSION: We propose that this interaction may be responsible for the Raf-induced inhibition of cytochrome c release from mitochondria in growth factor starved cells. Moreover, C-Raf kinase-induced VDAC inhibition may regulate the metabolic function of mitochondria and mediate the switch to aerobic glycolysis that is common to cancer cells

MYC is a metastasis gene for non-small-cell lung cancer.

Metastasis is a process by which cancer cells learn to form satellite tumors in distant organs and represents the principle cause of death of patients with solid tumors. NSCLC is the most lethal human cancer due to its high rate of metastasis. Lack of a suitable animal model has so far hampered analysis of metastatic progression. We have examined c-MYC for its ability to induce metastasis in a C-RAF-driven mouse model for non-small-cell lung cancer. c-MYC alone induced frank tumor growth only after long latency at which time secondary mutations in K-Ras or LKB1 were detected reminiscent of human NSCLC. Combination with C-RAF led to immediate acceleration of tumor growth, conversion to papillary epithelial cells and angiogenic switch induction. Moreover, addition of c-MYC was sufficient to induce macrometastasis in liver and lymph nodes with short latency associated with lineage switch events. Thus we have generated the first conditional model for metastasis of NSCLC and identified a gene, c-MYC that is able to orchestrate all steps of this process. Potential markers for detection of metastasis were identified and validated for diagnosis of human biopsies. These markers may represent targets for future therapeutic intervention as they include genes such as Gata4 that are exclusively expressed during lung development

Diverse effects of RacV12 on cell transformation by Raf: partial inhibition of morphological transformation versus deregulation of cell cycle control

AbstractActivated Raf kinases and Rac GTPases were shown to cooperate in the oncogenic transformation of fibroblasts, which is characterised by the disassembly of the cellular actin cytoskeleton, a nearly complete loss of focal adhesion complexes and deregulated cell proliferation. This is surprising since the Rac GTPase induces actin structures and the adhesion of suspended cells to extracellular matrix proteins. NIH 3T3 cells expressing a hydroxytamoxifen-inducible oncogenic c-Raf-1–oestrogen receptor fusion protein (c-Raf-1-BxB-ERTM, N-BxB-ERTM cells) undergo morphological transformation upon stimulation of the Raf kinase. We show that treatment with the Rac, Rho and Cdc42 activating Escherichia coli toxin CNF1 or coexpression of an activated RacV12 mutant partially inhibits and reverses the disassembly of cellular actin structures and focal adhesion complexes by oncogenic Raf. Activation of the Rac GTPase restores actin structures and focal adhesion complexes at the cellular boundary, leading to spreading of the otherwise spindle-shaped Raf-transformed cells. Actin stress fibres, however, which are regulated by the function of the Rho GTPase, are disassembled by oncogenic Raf even in the presence of activated Rac and Rho. With respect to the RacV12-mediated spreading of Raf-transformed cells, we postulate an anti-oncogenic function of the activated Rac. Another feature of cell transformation is the deregulation of cell cycle control. NIH 3T3 cells expressing high levels of the c-Raf-1-BxB-ERTM protein undergo a cell cycle arrest upon stimulation of the oncogenic Raf kinase. Our results show that in N-BxB-ERTM-RacV12 cells the expression of the activated RacV12 mediates cell proliferation in the presence of high-intensity Raf signals and high levels of the Cdk inhibitor p21Cip1. These results indicate a pro-oncogenic function of the Rac GTPase with respect to the deregulation of cell cycle control

Specific antibody-receptor interactions trigger InlAB-independent uptake of listeria monocytogenes into tumor cell lines

<p>Abstract</p> <p>Background</p> <p>Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient <it>Listeria monocytogenes </it>strain (Lm-spa⁺), which expresses protein A of <it>Staphylococcus aureus </it>(SPA) and anchors SPA in the correct orientation on the bacterial cell surface.</p> <p>Results</p> <p>This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin^®) or Cetuximab (Erbitux^®) to Lm-spa⁺, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlAB-independent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptor-mediated internalization of Lm-spa⁺is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface.</p> <p>Conclusions</p> <p>Binding of receptor-specific antibodies to SPA-expressing <it>L. monocytogenes </it>may represent a promising approach to target <it>L. monocytogenes </it>to host cells expressing specific receptors triggering internalization.</p

Shigella Mediated Depletion of Macrophages in a Murine Breast Cancer Model Is Associated with Tumor Regression

A tumor promoting role of macrophages has been described for a transgenic murine breast cancer model. In this model tumor-associated macrophages (TAMs) represent a major component of the leukocytic infiltrate and are associated with tumor progression. Shigella flexneri is a bacterial pathogen known to specificly induce apotosis in macrophages. To evaluate whether Shigella-induced removal of macrophages may be sufficient for achieving tumor regression we have developed an attenuated strain of S. flexneri (M90TΔaroA) and infected tumor bearing mice. Two mouse models were employed, xenotransplantation of a murine breast cancer cell line and spontanous breast cancer development in MMTV-HER2 transgenic mice. Quantitative analysis of bacterial tumor targeting demonstrated that attenuated, invasive Shigella flexneri primarily infected TAMs after systemic administration. A single i.v. injection of invasive M90TΔaroA resulted in caspase-1 dependent apoptosis of TAMs followed by a 74% reduction in tumors of transgenic MMTV-HER-2 mice 7 days post infection. TAM depletion was sustained and associated with complete tumor regression

Mitochondrial protein p26 BCL2 reduces growth factor requirements of NIH3T3 fibroblasts

The BCL2 (B cell lymphoma/leukemia-2) proto-oncogene encodes a 26-kDa protein that has been localized to the inner mitochondrial membrane and that has been shown to enhance the survival of some types of hematopoietic cells. Here we show that NIH3T3 fibroblasts stably transfected with a BCL2 expression plasmid exhibit reduced dependence on competence-inducing growth factors (platelet-derived growth factor, PDGF; epidermal growth factor, EGF) for initiation of DNA synthesis. The importance of BCL2 for growth factorinduced proliferation of these cells was further confirmed by the useage of BCL2 antisense oligodeoxynucleotides. The mechanisms by which overexpression of p26 BCL2 contributes to fibroblast proliferation are unknown, but do not involve alterations in: (a) the production of inositol triphosphates (IP3), (b) PDGF-induced transient elevations in cytosolic Ca2+ ions, or (c) the activity of protein kinase C enzymes in these transfected cells. The results imply that changes in mitochondrial functions play an important role in the early stages of the cell cycle that render 3T3 cells competent to respond to the serum progression factors that stimulate entry into S-phase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29195/1/0000249.pd

Electroweak Physics for Color Superconductivity

We construct the effective theories describing the electroweak interactions for the low energy excitations associated with the color superconductive phases of QCD at high matter density. The main result, for the 3 flavor case, is that the quasiparticle Goldstone boson $\pi^0$ decay into two physical massless photons is identical to the zero density case once we use the new Goldstone decay constant and the modified electric charge $\widetilde{e}=e \cos\theta$, with $\tan\theta =2e/\sqrt{3}g_s$ and $g_s$ the strong coupling constant. For 2 flavors we find that the coupling of the quarks to the neutral vector boson $Z^0$ is modified with respect to the zero density case. We finally point out possible applications of our result to the physics of compact objects.Comment: 23 pages, 1 Figure, RevTex. More discussion and references adde