SummaryRio1p was identified as a protein serine kinase founding a novel subfamily. It is highly conserved from Archaea to man and only distantly related to previously established protein kinase families. Nevertheless, analysis of multiple protein sequence alignments shows that those amino acid residues that are important for either structure or catalytic activity in conventional protein kinases are also conserved in members of the Rio1p family at the respective positions (corresponding to domains I-XI of protein kinases). Recombinant Rio1p from Escherichia coli and tagged Rio1p from yeast has kinase activity in vitro, and mutation of amino acid residues that are conserved and indispensable for catalytic activity (i.e. ATP-binding motif, catalytic centre) abrogates activity. RIO1 is essential in yeast and plays a role in cell cycle progression. After sporulation of RIO1/rio1 diploids, RIO1-disrupted progeny cease growth after one to three cell divisions and arrest as either large unbudded or large-budded cells. Cells deprived of Rio1p are enlarged and arrest either in G1 or in mitosis mainly with the DNA at the bud neck and short spindles (a phenotype also seen in cells carrying a weak allele), suggesting that Rio1p activity is required for at least at two steps during the cell division cycle: for entrance into S phase and for exit from mitosis. The weak RIO1 allele leads to increased plasmid loss. IntroductionProtein kinases are important regulators of a plethora of processes in eukaryotes. The cell division cycle is a Novel class of protein kinases 311The RIO1 gene encodes a protein of 484 amino acids (deduced molecular mass 56.0 kDa). It is transcribed constitutively at a very low level (Angermayr and Bandlow, 1997). Little is known about the biological role of Rio1p, and reports on its function are conflicting. In Emericella nidulans, a mutant orthologue of RIO1, sudD, has been identified as a suppressor of a mutant blocked in mitosis (bimD; Anaya et al., 1998). BimD is the orthologue of yeast Pds5p, a component of a macromolecular complex that holds sister chromatids together after replication until the onset of anaphase (Hartman et al., 2000), suggesting that Rio1p might be involved in the control of chromatid segregation during mitosis. Recently, Rio1p was isolated in a screen for mutants synthetically lethal with a mutant allele of GAR1, an essential gene involved in 18S rRNA maturation (Vanrobays et al., 2001). Depletion of Rio1p led to the accumulation of 20S prerRNA.We report here that Rio1p is a protein serine kinase. Although only distantly related to previously established protein kinase families, it displays the subdomain structure characteristic of protein kinases and has protein kinase activity in vitro. Some of the conserved residues are shown to be essential for viability and enzymatic activity. Thus, Rio1p constitutes the founding member of a novel subfamily of protein kinases. The protein is mainly located in the cytoplasm. It plays an important role in G1 to S transition and in the control ...

Acquired resistance to classical chemotherapeutics is a major obstacle in cancer treatment. Doxorubicin is frequently used in breast cancer therapy either as single-agent or in combination with other drugs like docetaxel and cyclophosphamide. All these chemotherapies have in common that they are administered sequentially and often result in chemoresistance. Here, we mimicked this pulse therapy of breast cancer patients in an in vitro cell culture model, where the epithelial breast cancer cell line BT474 was sequentially treated with doxorubicin for several treatment cycles. In consequence, we obtained chemoresistant cells displaying a mesenchymal-like phenotype with decreased levels of miR-200c. To investigate the involvement of miR-200c in resistance formation, we inhibited and overexpressed miR-200c in different cell lines. Thereby, the cells were rendered more resistant or susceptible to doxorubicin treatment. Moreover, the receptor tyrosine kinase TrkB and the transcriptional repressor Bmi1 were identified as miR-200c targets mediating the drug resistance. Hence, we provide a mechanism of acquired resistance to doxorubicin that is caused by the loss of miR-200c. Along with this, our study demonstrates the complex network of microRNA mediated chemoresistance highlighting the challenges in cancer therapy and the importance of novel microRNA-modulating anticancer agents.

Purpose: Establishment of antiapoptotic signaling pathways in tumor cells is a major cause for the failure of chemotherapy against cancer.Toinvestigate the underlying mechanisms, we developed an experimentalapproach that is basedonthe genetic plasticity of cancer cells and the selectionfor cell survival on treatment with chemotherapeutic agents. Experimental Design: Gene expression changes of surviving cell clones were analyzed by macroarrays. Involvement of fibroblast growth factor receptor 4 (FGFR4) in antiapoptotic pathways was elucidated by apoptosis assays, small interfering RNA experiments, and an antagonistic antibody. Results:We show that FGFR4 gene expression is up-regulated in doxorubicin-treated, apoptosisresistant cancer cell clones. Ectopic expression of FGFR4 in cancer cells led to reduced apoptosis sensitivity on treatment with doxorubicin or cyclophosphamide, whereas knockdown of endogenous FGFR4 expression in breast cancer cell lines had the opposite effect. FGFR4 overexpression resulted in Bcl-xl up-regulation at both mRNA and proteinlevels. Knockdown of FGFR4 expression by small interfering RNA caused a decrease in phospho-extracellular signal-regulated kinase 1/2 levels and reduced Bcl-xl expression. Moreover, an antagonistic FGFR4 antibody suppressed the resistance of cancer cells with endogenous FGFR4 expression against apoptosis-inducing chemotherapeutic agents. Conclusion: Based on these findings, we propose an antiapoptotic signaling pathway that is initiatedby FGFR4 andregulatingthe expressionof Bcl-xl throughthemitogen-activatedproteinkinase cascade. Our findings are exemplary for a novel strategy toward the elucidation of diverse signaling pathways that define antiapoptotic potential in cancer cells.These observations open new avenues toward the diagnosis of chemoresistant tumors and therapies targeting FGFR4-overexpressing cancers.Breast cancer is the most frequent malignancy among women in the western world. 3,4 Although much effort has been invested into designing new therapies, classic chemotherapeutic agents such as doxorubicin (Adriamycin) are still widely employed in the clinic (1). Chemotherapeutic drugs are used as primary or adjuvant therapy with response rates from 60% to 100% (2, 3). A major problem of most of the current therapies are the sometimes severe side effects and the intrinsic or acquired drug resistance of cancer cells to these drugs, which lead to relapse and metastatic progression of the tumor. This is reflected by the decline in the response rates with second-line chemotherapy to 20% to 30% (4).Motivated by these facts, a comprehensive characterization of genes contributing to a drug resistance phenotype has been done in this study. Based on an approach of Hudziak et al. (5) and Abraham et al. (6), where tumor necrosis factor-a-resistant and Fas ligand-resistant cell clones were established, we respectively took advantage of the genomic instability of breast cancer cell lines to generate doxorubicin-resistant cell clones. When treated with chemothera...

The GTPase K-ras is involved in a variety of cellular processes such as differentiation, proliferation and survival. However, activating mutations, which frequently occur in many types of cancer, turn KRAS into one of the most prominent oncogenes. Likewise, miR-200c is a key player in tumorigenesis functioning as a molecular switch between an epithelial, non-migratory, chemosensitive and a mesenchymal, migratory, chemoresistant state. While it has been reported that KRAS is modulated by several tumor suppressor miRNAs, this is the first report on the regulation of KRAS by miR-200c, both playing a pivotal role in oncogenesis. We show that KRAS is a predicted target of miR-200c and that the protein expression of KRAS inversely correlates with the miR-200c expression in a panel of human breast cancer cell lines. KRAS was experimentally validated as a target of miR-200c by Western blot analyses and luciferase reporter assays. Furthermore, the inhibitory rffect of miR-200c-dependent KRAS silencing on proliferation and cell cycle was demonstrated in dfferent breast and lung cancer cell lines. Thereby, the particular role of KRAS was dissected from the role of all the other miR-200c targets by specific knockdown experiments using siRNA against KRAS. Cell lines harboring an activating KRAS mutation were similarly affected by miR-200c as well as by the siRNA against KRAS. However, in a cell line with wild-type KRAS only miR-200c was able to change proliferation and cell cycle. Our findings suggest that miR-200c is a potent inhibitor of tumor progression and therapy resistance, by regulating a multitude of oncogenic pathways including the RAS pathway. Thus, miR-200c may cause stronger anti-tumor efffects than a specific siRNA against KRAS, emphasizing the potential role of miR-200c as tumor suppressive miRNA

The myxobacterial agent archazolid inhibits the vacuolar proton pump V-ATPase. V-ATPases are ubiquitously expressed ATPdependent proton pumps, which are known to regulate the pH in endomembrane systems and thus play a crucial role in endo-and exocytotic processes of the cell. As cancer cells depend on a highly active secretion of proteolytic proteins in order to invade tissue and form metastases, inhibition of V-ATPase is proposed to affect the secretion profile of cancer cells and thus potentially abrogate their metastatic properties. Archazolid is a novel V-ATPase inhibitor. Here, we show that the secretion pattern of archazolid treated cancer cells includes various prometastatic lysosomal proteins like cathepsin A, B, C, D and Z. In particular, archazolid induced the secretion of the proforms of cathepsin B and D. Archazolid treatment abrogates the cathepsin B maturation process leading to reduced intracellular mature cathepsin B protein abundance and finally decreased cathepsin B activity, by inhibiting mannose-6-phoshate receptor-dependent trafficking. Importantly, in vivo reduced cathepsin B protein as well as a decreased proteolytic cathepsin B activity was detected in tumor tissue of archazolid-treated mice. Our results show that inhibition of V-ATPase by archazolid reduces the activity of prometastatic proteases like cathepsin B in vitro and in vivo.Vacuolar (H1)-ATPases (V-ATPases) are found on various membranes, including lysosomes, endosomes, vesicles and the plasma membrane. As V-ATPases are ATP-dependent proton pumps they are crucial for maintaining the pH of these compartments. Thus, they play a vital role in various cellular processes, like intracellular targeting of lysosomal enzymes, protein processing and degradation as well as receptor-mediated endocytosis.