Structural changes in Mcm5 protein bypass Cdc7-Dbf4 function and reduce replication origin efficiency in Saccharomyces cerevisiae.

Eukaryotic chromosomal replication is a complicated process with many origins firing at different efficiencies and times during S phase. Prereplication complexes are assembled on all origins in G(1) phase, and yet only a subset of complexes is activated during S phase by DDK (for Dbf4-dependent kinase) (Cdc7-Dbf4). The yeast mcm5-bob1 (P83L) mutation bypasses DDK but results in reduced intrinsic firing efficiency at 11 endogenous origins and at origins located on minichromosomes. Origin efficiency may result from Mcm5 protein assuming an altered conformation, as predicted from the atomic structure of an archaeal MCM (for minichromosome maintenance) homologue. Similarly, an intragenic mutation in a residue predicted to interact with P83L suppresses the mcm5-bob1 bypass phenotype. We propose DDK phosphorylation of the MCM complex normally results in a single, highly active conformation of Mcm5, whereas the mcm5-bob1 mutation produces a number of conformations, only one of which is permissive for origin activation. Random adoption of these alternate states by the mcm5-bob1 protein can explain both how origin firing occurs independently of DDK and why origin efficiency is reduced. Because similar mutations in mcm2 and mcm4 cannot bypass DDK, Mcm5 protein may be a unique Mcm protein that is the final target of DDK regulation.

Crosstalk between invadopodia and the extracellular matrix.

The scaffold protein Tks5α is required for invadopodia-mediated cancer invasion both in vitro and in vivo. We have previously also revealed a role for Tks5 in tumor cell growth using three-dimensional (3D) culture model systems and mouse transplantation experiments. Here we use both 3D and high-density fibrillar collagen (HDFC) culture to demonstrate that native collagen-I, but not a form lacking the telopeptides, stimulated Tks5-dependent growth, which was dependent on the DDR collagen receptors. We used microenvironmental microarray (MEMA) technology to determine that laminin, fibronectin and tropoelastin also stimulated invadopodia formation. A Tks5α-specific monoclonal antibody revealed its expression both on microtubules and at invadopodia. High- and super-resolution microscopy of cells in and on collagen was then used to place Tks5α at the base of invadopodia, separated from much of the actin and cortactin, but coincident with both matrix metalloprotease and cathepsin proteolytic activity. Inhibition of the Src family kinases, cathepsins or metalloproteases all reduced invadopodia length but each had distinct effects on Tks5α localization. These studies highlight the crosstalk between invadopodia and extracellular matrix components, and reveal the invadopodium to be a spatially complex structure.

Interleukin-1beta and fibroblast growth factor receptor 1 cooperate to induce cyclooxygenase-2 during early mammary tumourigenesis.

INTRODUCTION: Inflammation within the tumour microenvironment correlates with increased invasiveness and poor prognosis in many types of cancer, including breast cancer. We have previously demonstrated that activation of a mouse mammary tumour virus (MMTV)-driven inducible fibroblast growth factor receptor 1 (iFGFR1) transgene in mammary epithelial cells results in an inflammatory response characterised by induction of inflammatory genes in the mammary gland. Specifically, we have observed increased levels of IL-1beta expression in the mammary gland following activation of iFGFR1 and have used the iFGFR1 model to elucidate the function of IL-1beta in promoting iFGFR1-induced mammary lesions. METHODS: To determine the functional consequences of IL-1beta induction during FGFR1-induced mammary tumourigenesis, the effects of IL-1beta inhibition on the formation of epithelial hyperplasias were examined using the MMTV-iFGFR1 transgenic mouse model. Further studies used a combination of the HC-11 mammary epithelial cell line that stably expresses iFGFR1 and the MMTV-iFGFR1 transgenic mice to further define the mechanisms of IL-1beta function. RESULTS: Inhibition of IL-1beta activity in vivo resulted in reduced iFGFR1-induced epithelial proliferation and formation of hyperplastic structures. Further studies demonstrated that treatment of mammary epithelial cells with IL-1beta-induced expression of cyclooxygenase (Cox)-2 both in vitro and in vivo. Finally, inhibition of Cox-2 prior to activation of iFGFR1 in the transgenic mice also resulted in decreased iFGFR1-induced formation of hyperplastic structures. CONCLUSIONS: The results from these studies indicate that targeting the inflammatory cytokine IL-1beta partially inhibits iFGFR1-induced formation of early-stage mammary lesions, in part through induction of Cox-2. These findings demonstrate that activation of a growth factor receptor in mammary epithelial cells results in increased expression of inflammatory mediators, which cooperate to promote the initiation of hyperplastic lesions in the mammary gland.

Functional conservation of beta-hairpin DNA binding domains in the Mcm protein of Methanobacterium thermoautotrophicum and the Mcm5 protein of Saccharomyces cerevisiae.

Mcm proteins are an important family of evolutionarily conserved helicases required for DNA replication in eukaryotes. The eukaryotic Mcm complex consists of six paralogs that form a heterohexameric ring. Because the intact Mcm2-7 hexamer is inactive in vitro, it has been difficult to determine the precise function of the different subunits. The solved atomic structure of an archaeal minichromosome maintenance (MCM) homolog provides insight into the function of eukaryotic Mcm proteins. The N-terminal positively charged central channel in the archaeal molecule consists of beta-hairpin domains essential for DNA binding in vitro. Eukaryotic Mcm proteins also have beta-hairpin domains, but their function is unknown. With the archaeal atomic structure as a guide, yeast molecular genetics was used to query the function of the beta-hairpin domains in vivo. A yeast mcm5 mutant with beta-hairpin mutations displays defects in the G1/S transition of the cell cycle, the initiation phase of DNA replication, and in the binding of the entire Mcm2-7 complex to replication origins. A similar mcm4 mutation is synthetically lethal with the mcm5 mutation. Therefore, in addition to its known regulatory role, Mcm5 protein has a positive role in origin binding, which requires coordination by all six Mcm2-7 subunits in the hexamer.

Activation of the FGFR-STAT3 pathway in breast cancer cells induces a hyaluronan-rich microenvironment that licenses tumor formation.

Aberrant activation of fibroblast growth factor receptors (FGFR) contributes to breast cancer growth, progression, and therapeutic resistance. Because of the complex nature of the FGF/FGFR axis, and the numerous effects of FGFR activation on tumor cells and the surrounding microenvironment, the specific mechanisms through which aberrant FGFR activity contributes to breast cancer are not completely understood. We show here that FGFR activation induces accumulation of hyaluronan within the extracellular matrix and that blocking hyaluronan synthesis decreases proliferation, migration, and therapeutic resistance. Furthermore, FGFR-mediated hyaluronan accumulation requires activation of the STAT3 pathway, which regulates expression of hyaluronan synthase 2 (HAS2) and subsequent hyaluronan synthesis. Using a novel in vivo model of FGFR-dependent tumor growth, we demonstrate that STAT3 inhibition decreases both FGFR-driven tumor growth and hyaluronan levels within the tumor. Finally, our results suggest that combinatorial therapies inhibiting both FGFR activity and hyaluronan synthesis is more effective than targeting either pathway alone and may be a relevant therapeutic approach for breast cancers associated with high levels of FGFR activity. In conclusion, these studies indicate a novel targetable mechanism through which FGFR activation in breast cancer cells induces a protumorigenic microenvironment.

Functional integration of acute myeloid leukemia into the vascular niche.

Vascular endothelial cells are a critical component of the hematopoietic microenvironment that regulates blood cell production. Recent studies suggest the existence of functional cross-talk between hematologic malignancies and vascular endothelium. Here we show that human acute myeloid leukemia (AML) localizes to the vasculature in both patients and in a xenograft model. A significant number of vascular tissue-associated AML cells (V-AML) integrate into vasculature in vivo and can fuse with endothelial cells. V-AML cells acquire several endothelial cell-like characteristics, including the upregulation of CD105, a receptor associated with activated endothelium. Remarkably, endothelial-integrated V-AML shows an almost fourfold reduction in proliferative activity compared with non-vascular-associated AML. Primary AML cells can be induced to downregulate the expression of their hematopoietic markers in vitro and differentiate into phenotypically and functionally defined endothelial-like cells. After transplantation, these leukemia-derived endothelial cells are capable of giving rise to AML. These novel functional interactions between AML cells and normal endothelium along with the reversible endothelial cell potential of AML suggest that vascular endothelium may serve as a previously unrecognized reservoir for AML.

Endothelial cells mitigate DNA damage and promote the regeneration of hematopoietic stem cells after radiation injury.

Endothelial cells (ECs) are an essential component of the hematopoietic microenvironment, which maintains and regulates hematopoietic stem cells (HSCs). Although ECs can support the regeneration of otherwise lethally-irradiated HSCs, the mechanisms are not well understood. To further understand this phenomenon, we studied HSC regeneration from irradiated bone marrow using co-culture with human aortic ECs (HAECs). Co-culture with HAECs induced a 24-fold expansion of long-term HSCs (CD150(+), lineage(lo), Sca-1(+), c-Kit(+); CD150(+)LSK cells) in vitro. These cells gave rise to functional hematopoietic stem and progenitor cells (HSPCs) with colony-forming activity, multilineage reconstitution and serial transplantation potential. Furthermore, HAECs significantly reduced DNA damage in irradiated LSK cells within 24h. Remarkably, we were able to delay the exposure of irradiated bone marrow to the regenerative, HAEC-derived signals for up to 48h and still rescue functional HSCs. G-CSF is the gold standard for promoting hematopoietic regeneration in vivo. However, when compared to HAECs, in vitro G-CSF treatment promoted lineage differentiation and regenerated 5-fold fewer CD150(+)LSK cells. Together, our results show that HAECs are powerful, direct mitigators of HSC injury and DNA damage. Identification of the HAEC-derived factors that rescue HSCs may lead to improved therapies for hematopoietic regeneration after radiation injury.

The structure and function of MCM from archaeal M. Thermoautotrophicum.

Eukaryotic chromosomal DNA is licensed for replication precisely once in each cell cycle. The mini-chromosome maintenance (MCM) complex plays a role in this replication licensing. We have determined the structure of a fragment of MCM from Methanobacterium thermoautotrophicum (mtMCM), a model system for eukaryotic MCM. The structure reveals a novel dodecameric architecture with a remarkably long central channel. The channel surface has an unusually high positive charge and binds DNA. We also show that the structure of the N-terminal fragment is conserved for all MCMs proteins despite highly divergent sequences, suggesting a common architecture for a similar task: gripping/remodeling DNA and regulating MCM activity. An mtMCM mutant protein equivalent to a yeast MCM5 (CDC46) protein with the bob1 mutation at its N terminus has only subtle structural changes, suggesting a Cdc7-bypass mechanism by Bob1 in yeast. Yeast bypass experiments using MCM5 mutant proteins support the hypothesis for the bypass mechanism.