Jirina Prochazkova

Receptor tyrosine kinases activate canonical WNT/β-catenin signaling via MAP kinase/LRP6 pathway and direct β-catenin phosphorylation.

Receptor tyrosine kinase signaling cooperates with WNT/β-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/β-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate β-catenin at Tyr142, which is known to increase cytoplasmic β-catenin concentration via release of β-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct β-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling.

Molecular pathology of the fibroblast growth factor family

The human fibroblast growth factor (FGF) family contains 22 proteins that regulate a plethora of physiological processes in both developing and adult organism. The mutations in the FGF genes were not known to play role in human disease until the year 2000, when mutations in FGF23 were found to cause hypophosphatemic rickets. Nine years later, seven FGFs have been associated with human disorders. These include FGF3 in Michel aplasia; FGF8 in cleft lip/palate and in hypogonadotropic hypogonadism; FGF9 in carcinoma; FGF10 in the lacrimal/salivary glands aplasia, and lacrimo‐auriculo‐dento‐digital syndrome; FGF14 in spinocerebellar ataxia; FGF20 in Parkinson disease; and FGF23 in tumoral calcinosis and hypophosphatemic rickets. The heterogeneity in the functional consequences of FGF mutations, the modes of inheritance, pattern of involved tissues/organs, and effects in different developmental stages provide fascinating insights into the physiology of the FGF signaling system. We review the current knowledge about the molecular pathology of the FGF family. Hum Mutat 30:1–11, 2009.

FGFR3 signaling induces a reversible senescence phenotype in chondrocytes similar to oncogene-induced premature senescence

Oncogenic activation of the RAS-ERK MAP kinase signaling pathway can lead to uncontrolled proliferation but can also result in apoptosis or premature cellular senescence, both regarded as natural protective barriers to cell immortalization and transformation. In FGFR3-related skeletal dyplasias, oncogenic mutations in the FGFR3 receptor tyrosine kinase cause profound inhibition of cartilage growth resulting in severe dwarfism, although many of the precise mechanisms of FGFR3 action remain unclear. Mutated FGFR3 induces constitutive activation of the ERK pathway in chondrocytes and, remarkably, can also cause both increased proliferation and apoptosis in growing cartilage, depending on the gestational age. Here, we demonstrate that FGFR3 signaling is also capable of inducing premature senescence in chondrocytes, manifested as reversible, ERK-dependent growth arrest accompanied by alteration of cellular shape, loss of the extracellular matrix, upregulation of senescence markers (alpha-GLUCOSIDASE, FIBRONECTIN, CAVEOLIN 1, LAMIN A, SM22alpha and TIMP 1), and induction of senescence-associated beta-GALACTOSIDASE activity. Our data support a model whereby FGFR3 signaling inhibits cartilage growth via exploiting cellular responses originally designed to eliminate cells harboring activated oncogenes.

NF449 is a novel inhibitor of fibroblast growth factor receptor 3 (FGFR3) signaling active in chondrocytes and multiple myeloma cells

The FGFR3 receptor tyrosine kinase represents an attractive target for therapy due to its role in several human disorders, including skeletal dysplasias, multiple myeloma, and cervical and bladder carcinomas. By using molecular library screening, we identified a compound named NF449 with inhibitory activity toward FGFR3 signaling. In cultured chondrocytes and murine limb organ culture, NF449 rescued FGFR3-mediated extracellular matrix loss and growth inhibition, which represent two major cellular phenotypes of aberrant FGFR3 signaling in cartilage. Similarly, NF449 antagonized FGFR3 action in the multiple myeloma cell lines OPM2 and KMS11, as evidenced by NF449-mediated reversal of ERK MAPK activation and transcript accumulation of CCL3 and CCL4 chemokines, both of which are induced by FGFR3 activation. In cell-free kinase assays, NF449 inhibited the kinase activity of both wild type and a disease-associated FGFR3 mutant (K650E) in a fashion that appeared non-competitive with ATP. Our data identify NF449 as a novel antagonist of FGFR3 signaling, useful for FGFR3 inhibition alone or in combination with inhibitors that target the ATP binding site.

The PI3K/AKT signaling pathway controls the quiescence of the low-Rhodamine123-retention cell compartment enriched for melanoma stem cell activity.

Melanoma is one of the most aggressive and extremely resistant to conventional therapies neoplasms. Recently, cellular resistance was linked to the cancer stem cell phenotype, still controversial and not well‐defined. In this study, we used a Rhodamine 123 (Rh123) exclusion assay to functionally identify stem‐like cells in metastatic human melanomas and melanoma cell lines. We demonstrate that a small subset of Rh123‐low‐retention (Rh123low) cells is enriched for stem cell‐like activities, including the ability to self‐renew and produce nonstem Rh123high progeny and to form melanospheres, recapitulating the phenotypic profile of the parental tumor. Rh123low cells are relatively quiescent and chemoresistant. At the molecular level, we show that melanoma Rh123low cells overexpress HIF1α, pluripotency factor OCT4, and the ABCB5 marker of melanoma stem cells and downregulate the expression of Cyclin D1 and CDK4. Interestingly, a short treatment with LY294002, an inhibitor of the PI3K/AKT pathway, specifically reverts a subset of Rh123high cells to the Rh123low phenotype, whereas treatment with inhibitors of mammalian target of rapamycin, phosphatase and tensin homolog or mitogen‐activated protein kinase signaling does not. This phenotypic switching was associated with reduced levels of the HIF1α transcript and an increase in the level of phosphorylated nuclear FOXO3a preferentially in Rh123low cells. Moreover, the Rh123low cells became less quiescent and displayed a significant increase in their melanosphere‐forming ability. All the above indicates that the Rh123low melanoma stem cell pool is composed of cycling and quiescent cells and that the PI3K/AKT signaling while maintaining the quiescence of Rh123low G0 cells promotes the exit of cycling cells from the stem cell compartment. STEM CELLS 2013;31:641–651

Fibroblast growth factor inhibits interferon γ-STAT1 and interleukin 6-STAT3 signaling in chondrocytes

Activation of fibroblast growth factor receptor 3 (FGFR3) leads to attenuation of cartilage growth. The members of the STAT family of transcription factors are believed to participate in FGFR3 signaling in cartilage, however the molecular mechanism of this action is poorly understood. Here, we demonstrate that a chronic FGF stimulus leads to accumulation of STAT1, 3, 5 and 6, evident in both in vitro chondrocyte model and murine limb explant cultures. Despite the accumulation, both endogenous and cytokine-induced activation of STAT1 and STAT3 is impaired by FGF, as demonstrated by imaging of active STAT nuclear translocation and analyses of STAT activatory phosphorylation and transcriptional activation. Further, we demonstrate that FGF induces expression of CIS, SOCS1 and SOCS3 inhibitors of gp130, a common receptor for the IL6-family of cytokines. Since cytokine-gp130 signaling represents an important positive regulator of cartilage, its inhibition may contribute to the growth-inhibitory effect of FGFR3 in cartilage.

Clinical and laboratory features of leukemias at the time of diagnosis: An analysis of 1,004 consecutive patients

To determine the major presenting features of the most frequent leukemias and to describe the period from the first symptoms to the final diagnosis, we analyzed the records of 1,004 consecutive patients with acute (AL) and chronic (CL) leukemias diagnosed between 2004 and 2009. In 24% of AL and 63% of CL, the diagnosis was made incidentally. In contrast to the frequency of the symptoms, their spectrum did not differ from historical studies, and fatigue and weakness were the most frequent symptoms observed [with the exception of acute promyelocytic leukemia (APL) where bleeding predominated]. More profound cytopenia was found in AL compared to CL. The median time from the first symptoms to final diagnosis was 3 weeks in AL and 4 weeks in CL. APL had the shortest time to a definitive diagnosis (2 weeks); however, a very short duration of symptoms was also observed in CML (3 weeks). This study is the largest series reported to date on symptoms in leukemia patients at the time of diagnosis, and the first such report that reflects on clinical practice in the new millennium.

Insulin resistance is an underlying mechanism of impaired glucose metabolism during nilotinib therapy: Correspondence

Impaired glucose metabolism (IGM) with hyperglycemia represents one of the most frequently observed adverse events (AE) during nilotinib therapy of chronic myeloid leukemia (CML). The exact mechanism of IGM remains controversial. Although a case report has shown a decrease in insulin secretion1 , our previous pilot data suggested development of insulin resistance as a possible mechanism.2 In this prospective study we aimed to confirm results from our pilot study using a larger cohort of CML patients treated with nilotinib and to compare results with data obtained on control groups receiving imatinib and dasatinib.