Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic ...landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases. No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes. MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The mitogen-activated protein kinase (MAPK) signaling pathways play essential roles in cell proliferation and differentiation. Recent studies also show the activation of MAPK signaling pathways in ...tumorigenesis, metastasis, and angiogenesis of multiple human malignancies, including renal cell carcinoma (RCC). To assess the role of this pathway in regulating the proliferation and survival of RCC cells, we first examined the expression of MAPK kinase (MKK) and MAPK in clear cell RCC and confirmed the overexpression of MKK1 and extracellular signal-regulated kinase 2 (ERK2) in these tumors. We then tested the effects of pharmacologic inhibition of MKK on human RCC cell lines, both in vitro and in vivo, using anthrax lethal toxin (LeTx), which cleaves and inactivates several MKKs. Western blotting showed that the phosphorylation levels of ERK, c-Jun-NH(2) kinase, and p38 MAPK decreased after 72 h of LeTx treatment. Exposure to LeTx for 72 h reduced cell proliferation by 20% without significant effects on cell cycle distribution and apoptosis. Anchorage-independent growth of RCC cells was dramatically inhibited by LeTx. In vivo studies showed that tumor growth of RCC xenografts could be suppressed by LeTx. Extensive necrosis and decreased tumor neovascularization were observed after LeTx treatment. LeTx also showed direct inhibition of proliferation of endothelial cells in vitro. Our results suggest that suppression of one or more MAPK signaling pathways may inhibit RCC growth through the disruption of tumor vasculature.
To identify inflammatory cytokines significantly elevated and independent of VEGF levels in the vitreous of proliferative diabetic retinopathy (PDR) patients that may serve as novel diagnostic ...factors or therapeutic targets.
Thirty-nine cytokines and chemokines were measured from the vitreous of 72 patients undergoing vitrectomy (29 controls and 43 PDR) via a magnetic bead-based immunoassay. Patient information, including sex, age, history of smoking, cancer diagnosis and treatment, and presence of diabetes and hypertension were also collected. Univariate and multivariate logistic regression analyses were performed to assess the association of cytokine concentrations and patient demographics with disease.
Nineteen cytokines were significantly elevated in the vitreous of PDR patients compared with controls, including five novel cytokines that have not previously been associated with PDR: sCD40L, GM-CSF, IFNα2, IL-12p40, and MCP-3. Sixteen cytokines were found to be statistically independent of VEGF. Of these, 14 show a statistically significant interaction with VEGF, while two do not. With regards to patient demographics, age and hypertension were statistically significant risk factors with the odds of disease decreasing with increasing age and increasing 3-fold for hypertensive patients.
This is the first report of a comprehensive multiplex analysis to identify novel VEGF-independent cytokines associated with PDR. Of the 39 inflammatory cytokines tested, 16 are predictive of disease risk, independent of VEGF levels. These PDR-associated cytokines represent potential targets in the treatment of PDR, both in conjunction with anti-VEGF therapy, as well as for patients that are nonresponders to such therapy.
MEK genomics in development and disease Bromberg-White, Jennifer L; Andersen, Nicholas J; Duesbery, Nicholas S
Briefings in functional genomics,
07/2012, Letnik:
11, Številka:
4
Journal Article
Odprti dostop
The mitogen-activated protein kinase kinases (the MAPK/ERK kinases; MKKs or MEKs) and their downstream substrates, the extracellular-regulated kinases have been intensively studied for their roles in ...development and disease. Until recently, it had been assumed any mutation affecting their function would have lethal consequences. However, the identification of MEK1 and MEK2 mutations in developmental syndromes as well as chemotherapy-resistant tumors, and the discovery of genomic variants in MEK1 and MEK2 have led to the realization the extent of genomic variation associated with MEKs is much greater than had been appreciated. In this review, we will discuss these recent advances, relating them to what is currently understood about the structure and function of MEKs, and describe how they change our understanding of the role of MEKs in development and disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Angiosarcoma is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. As a model for human angiosarcoma, we studied primary cells and tumorgrafts ...derived from canine hemangiosarcoma (HSA), which is also an endothelial malignancy with similar presentation and histology. Primary cells isolated from HSA showed constitutive extracellular signal-regulated kinase (ERK) activation. The mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor CI-1040 reduced ERK activation and the viability of primary cells derived from visceral, cutaneous, and cardiac HSA in vitro. HSA-derived primary cells were also sensitive to sorafenib, an inhibitor of B-Raf and multireceptor tyrosine kinases. In vivo, CI-1040 or PD0325901 decreased the growth of cutaneous cell-derived xenografts and cardiac-derived tumorgrafts. Sorafenib decreased tumor size in both in vivo models, although cardiac tumorgrafts were more sensitive. In human angiosarcoma, we noted that 50% of tumors stained positively for phosphorylated ERK1/2 and that the expression of several MEK-responsive transcription factors was upregulated. Our data showed that MEK signaling is essential for the growth of HSA in vitro and in vivo and provided evidence that the same pathways are activated in human angiosarcoma. This indicates that MEK inhibitors may form part of an effective therapeutic strategy for the treatment of canine HSA or human angiosarcoma, and it highlights the use of spontaneous canine cancers as a model of human disease.
Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a ...metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.
Angiosarcoma (AS) is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. Using tumorgraft models, we previously showed that AS is sensitive to ...small-molecule inhibitors that target mitogen-activated/extracellular-signal-regulated protein kinase kinases 1 and 2 (MEK). The objective of this study was to identify drugs that combine with MEK inhibitors to more effectively inhibit AS growth. We examined the in vitro synergy between the MEK inhibitor PD0325901 and inhibitors of eleven common cancer pathways in melanoma cell lines and canine angiosarcoma cell isolates. Combination indices were calculated using the Chou-Talalay method. Optimized combination therapies were evaluated in vivo for toxicity and efficacy using canine angiosarcoma tumorgrafts. Among the drugs we tested, rapamycin stood out because it showed strong synergy with PD0325901 at nanomolar concentrations. We observed that angiosarcomas are insensitive to mTOR inhibition. However, treatment with nanomolar levels of mTOR inhibitor renders these cells as sensitive to MEK inhibition as a melanoma cell line with mutant BRAF. Similar results were observed in B-Raf wild-type melanoma cells as well as in vivo, where treatment of canine AS tumorgrafts with MEK and mTOR inhibitors was more effective than monotherapy. Our data show that a low dose of an mTOR inhibitor can dramatically enhance angiosarcoma and melanoma response to MEK inhibition, potentially widening the field of applications for MEK-targeted therapy.
Anthrax lethal toxin produced by the bacterium Bacillus anthracis is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), inactivates
members of ...the mitogen-activated protein kinase kinase or MEK family through proteolysis of their NH 2 termini. However, neither the substrate requirements for LF cleavage nor the mechanism by which proteolysis inactivates MEK
have been demonstrated. By means of deletion mutant analysis and site-directed mutagenesis, we have identified an LFIR ( LF
i nteracting r egion) in the COOH-terminal kinase domain of MEK1 adjacent to the proline-rich region, which is essential for LF-mediated
proteolysis of MEK. Point mutations in this region block proteolysis but do not alter the kinase activity of MEK. Similar
mutations in MEK6 also prevent proteolysis, indicating that this region is functionally conserved among MEKs. In addition,
NH 2 -terminal proteolysis of MEK1 by LF was found to reduce not only the affinity of MEK1 for its substrate mitogen-activated
protein kinase but also its intrinsic kinase activity, indicating that the NH 2 -terminal end of MEK is important not only for substrate interaction but also for catalytic activity.
Mitogen-activated protein kinase kinases (MKK or MEK) 1 and 2 are usually treated as redundant kinases. However, in assessing their relative contribution towards ERK-mediated biologic response ...investigators have relied on tests of necessity, not sufficiency. In response we developed a novel experimental model using lethal toxin (LeTx), an anthrax toxin-derived pan-MKK protease, and genetically engineered protease resistant MKK mutants (MKKcr) to test the sufficiency of MEK signaling in melanoma SK-MEL-28 cells. Surprisingly, ERK activity persisted in LeTx-treated cells expressing MEK2cr but not MEK1cr. Microarray analysis revealed non-overlapping downstream transcriptional targets of MEK1 and MEK2, and indicated a substantial rescue effect of MEK2cr on proliferation pathways. Furthermore, LeTx efficiently inhibited the cell proliferation and anchorage-independent growth of SK-MEL-28 cells expressing MKK1cr but not MEK2cr. These results indicate in SK-MEL-28 cells MEK1 and MEK2 signaling pathways are not redundant and interchangeable for cell proliferation. We conclude that in the absence of other MKK, MEK2 is sufficient for SK-MEL-28 cell proliferation. MEK1 conditionally compensates for loss of MEK2 only in the presence of other MKK.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
