Patrick G. Telmer

Insights into the Conformational Equilibria of Maltose-binding Protein by Analysis of High Affinity Mutants.

The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft. The wild-type protein has a KD of 1200 nm for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nm; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nm. Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nm for maltotriose. Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity. Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose. The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP. These studies show that interactions in the interface opposite the ligand binding cleft, which we term the “balancing interface,” are responsible for modulating the affinity of MBP for its ligand. Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation.

RHAMM promotes interphase microtubule instability and mitotic spindle integrity through MEK1/ERK1/2 activity.

An oncogenic form of RHAMM (receptor for hyaluronan-mediated motility, mouse, amino acids 163–794 termed RHAMMΔ163) is a cell surface hyaluronan receptor and mitotic spindle protein that is highly expressed in aggressive human cancers. Its regulation of mitotic spindle integrity is thought to contribute to tumor progression, but the molecular mechanisms underlying this function have not previously been defined. Here, we report that intracellular RHAMMΔ163 modifies the stability of interphase and mitotic spindle microtubules through ERK1/2 activity. RHAMM−/− mouse embryonic fibroblasts exhibit strongly acetylated interphase microtubules, multi-pole mitotic spindles, aberrant chromosome segregation, and inappropriate cytokinesis during mitosis. These defects are rescued by either expression of RHAMM or mutant active MEK1. Mutational analyses show that RHAMMΔ163 binds to α- and β-tubulin protein via a carboxyl-terminal leucine zipper, but in vitro analyses indicate this interaction does not directly contribute to tubulin polymerization/stability. Co-immunoprecipitation and pulldown assays reveal complexes of RHAMMΔ163, ERK1/2-MEK1, and α- and β-tubulin and demonstrate direct binding of RHAMMΔ163 to ERK1 via a D-site motif. In vitro kinase analyses, expression of mutant RHAMMΔ163 defective in ERK1 binding in mouse embryonic fibroblasts, and blocking MEK1 activity collectively confirm that the effect of RHAMMΔ163 on interphase and mitotic spindle microtubules is mediated by ERK1/2 activity. Our results suggest a model wherein intracellular RHAMMΔ163 functions as an adaptor protein to control microtubule polymerization during interphase and mitosis as a result of localizing ERK1/2-MEK1 complexes to their tubulin-associated substrates.

Hyaluronan, Inflammation, and Breast Cancer Progression

Breast cancer-induced inflammation in the tumor reactive stroma supports invasion and malignant progression and is contributed to by a variety of host cells including macrophages and fibroblasts. Inflammation appears to be initiated by tumor cells and surrounding host fibroblasts that secrete pro-inflammatory cytokines and chemokines and remodel the extracellular matrix (ECM) to create a pro-inflammatory “cancerized” or tumor reactive microenvironment that supports tumor expansion and invasion. The tissue polysaccharide hyaluronan (HA) is an example of an ECM component within the cancerized microenvironment that promotes breast cancer progression. Like many ECM molecules, the function of native high-molecular weight HA is altered by fragmentation, which is promoted by oxygen/nitrogen free radicals and release of hyaluronidases within the tumor microenvironment. HA fragments are pro-inflammatory and activate signaling pathways that promote survival, migration, and invasion within both tumor and host cells through binding to HA receptors such as CD44 and RHAMM/HMMR. In breast cancer, elevated HA in the peri-tumor stroma and increased HA receptor expression are prognostic for poor outcome and are associated with disease recurrence. This review addresses the critical issues regarding tumor-induced inflammation and its role in breast cancer progression focusing specifically on the changes in HA metabolism within tumor reactive stroma as a key factor in malignant progression.

Specific Sizes of Hyaluronan Oligosaccharides Stimulate Fibroblast Migration and Excisional Wound Repair

The extracellular matrix polysaccharide hyaluronan (HA) plays a key role in both fibrotic and regenerative tissue repair. Accumulation of high molecular weight HA is typical of regenerative repair, which is associated with minimal inflammation and fibrosis, while fragmentation of HA is typical of postnatal wounds, which heal in the presence of inflammation and transient fibrosis. It is generally considered that HA oligosaccharides and fragments of a wide size range support these processes of adult, fibrotic wound repair yet the consequences of sized HA fragments/oligosaccharides to each repair stage is not well characterized. Here, we compared the effects of native HA, HA oligosaccharide mixtures and individual sizes (4–10mer oligosaccharides, 5 and, 40 kDa) of HA oligosaccharides and fragments, on fibroblast migration in scratch wound assays and on excisional skin wound repair in vivo. We confirm that 4–10mer mixtures significantly stimulated scratch wound repair and further report that only the 6 and 8mer oligosaccharides in this mixture are responsible for this effect. The HA 6mer promoted wound closure, accumulation of wound M1 and M2 macrophages and the M2 cytokine TGFβ1, but did not increase myofibroblast differentiation. The effect of 6mer HA on wound closure required both RHAMM and CD44 expression. In contrast, The 40 kDa HA fragment inhibited wound closure, increased the number of wound macrophages but had no effect on TGFβ1 accumulation or subsequent fibrosis. These results show that specific sizes of HA polymer have unique effects on postnatal wound repair. The ability of 6mer HA to promote wound closure and inflammation resolution without increased myofibroblast differentiation suggests that this HA oligosaccharide could be useful for treatment of delayed or inefficient wound repair where minimal fibrosis is advantageous.

How does a protein with dual mitotic spindle and extracellular matrix receptor functions affect tumor susceptibility and progression

The mechanisms responsible for the oncogenic effects of the hyaluronan (HA) receptor and mitotic spindle binding protein, RHAMM, are poorly understood. On one hand, extracellular RHAMM interacts with HA and cell-surface receptors such as CD44 to coordinately activate the MAPK/ERK1,2 pathway, thus contributing to the spread and proliferation of tumor cells. On the other hand, intracellular RHAMM decorates mitotic spindles and is necessary for spindle formation and progression through G2/M and overexpression or loss of RHAMM can result in multi-pole spindles and chromosome mis-segregation. The deregulation of these intracellular functions could lead to genomic instability and fuel tumor progression. This suggests that both extracellular and intracellular RHAMM can promote tumor progression. Intracellular RHAMM can bind directly to ERK1 to form complexes with ERK2, MEK1 and ERK1,2 substrates, and we present a model whereby RHAMM’s function is as a scaffold protein, controlling activation and targeting of ERK1,2 to specific substrates.

Stimulation of the maltose transporter ATPase by unliganded maltose binding protein

ATP hydrolysis by the maltose transporter (MalFGK(2)) is regulated by maltose binding protein (MBP). Binding of maltose to MBP brings about a conformational change from open to closed that leads to a strong stimulation of the MalFGK(2) ATPase. In this study, we address the long-standing but enigmatic observation that unliganded MBP is also able to stimulate MalFGK(2). Although the mechanism of this stimulation is not understood, it is sometimes attributed to a small amount of closed (but unliganded) MBP that may exist in solution. To gain insight into how MBP regulates the MalFGK(2) ATPase, we have investigated whether the open or the closed conformation of MBP is responsible for MalFGK(2) stimulation in the absence of maltose. The effect of MBP concentration on the stimulation of MalFGK(2) was assessed: for unliganded MBP, the apparent K(M) for stimulation of MalFGK(2) was below 1 microM, while for maltose-bound MBP, the K(M) was approximately 15 microM. We show that engineered MBP molecules in which the open-closed equilibrium has been shifted toward the closed conformation have a decreased ability to stimulate MalFGK(2). These results indicate that stimulation of the MalFGK(2) ATPase by unliganded MBP does not proceed through a closed conformation and instead must operate through a different mechanism than stimulation by liganded MBP. One possible explanation is that the open conformation is able to activate the MalFGK(2) ATPase directly.

Hyaluronan Associated Inflammation and Microenvironment Remodelling Influences Breast Cancer Progression

Caitlin Ward1, Catalina Vasquez1,2, Cornelia Tolg1,3, Patrick G. Telmer1 and Eva Turley1,4,5 1London Regional Cancer Program, London Health Sciences Center, Victoria Hospital, London ON 2Dept. of Medical Biophysics University of Western Ontario 3The Hospital for Sick Children, Toronto ON 4Dept. of Oncology, University of Western Ontario, London ON 5Dept. of Biochemistry, University of Western Ontario, London ON Canada

Translational models of prostate cancer bone metastasis

Metastatic disease is the principal cause of prostate-cancer-related mortality. Our ability to accurately recapitulate the spread of prostate cancer to bone — the most common site of metastasis — is critical to the development of novel metastasis-directed therapies. Several translational models of prostate cancer bone metastasis have been developed, including animal models, cell line injection models, 3D in vitro models, bone implant models, and patient-derived xenograft models. The use of these models has led to numerous advances in elucidating the molecular mechanisms of metastasis and innovations in targeted therapy. Despite this progress, current models are limited by a failure to holistically reproduce each individual element of the metastatic cascade in prostate cancer bone metastasis. In addition, factors such as accurate recapitulation of immunobiological events and improvements in tumour heterogeneity require further consideration. Knowledge gained from historical and currently used models will improve the development of next-generation models. An introspective appraisal of current preclinical models demonstrating bone metastases is warranted to narrow research focus, improve future translational modelling, and expedite the delivery of urgently needed metastasis-directed treatments.In this Review, Berish and colleagues outline the development of currently used models of prostate cancer bone metastasis and discuss mechanistic and therapeutic advances made using these models. The authors also suggest future directions to improve the applicability of these models to the metastatic cascade and human disease.Key pointsThe development of novel metastasis-directed prostate cancer therapies is highly reliant on our ability to accurately reproduce the underlying mechanisms in vivo.Existing models frequently employ a modular approach towards recapitulating particular aspects of disease progression.Each model possesses specific advantages and limitations that are important to experimental design and outcomes.Several valuable molecular and therapeutic advances have been made, despite the potential limitations of current models.The aims of next-generation models should be to improve tumour heterogeneity and enable the study of disease immunobiology.

Abstract 1162: Fibroblast RHAMM promotes breast cancer aggression by promoting expression of a subset of ERK1,2 target genes

The microenvironment plays a key role in cancer progression and metabolism of the extracellular matrix component hyaluronan (HA), a glycosaminoglycan, is associated with breast tumor progression. High molecular weight HA is degraded by hyaluronidases and free radicals to fragments of heterogeneous size, which accumulate in the peritumor stroma and activate signaling pathways in both tumor and stromal cells. The HA receptor RHAMM is a multifunctional protein that is found on the cell surface, inside the nucleus and the mitotic spindles of mesenchymal cells. RHAMM expression and HA accumulation in the tumor stroma are linked to breast tumor progression, predicting that RHAMM/HA regulated signaling is key to breast cancer progression. An N-terminally truncated RHAMM isoform, RHAMMonc, is oncogenic when overexpressed in mesenchymal cells. Intracellular RHAMMonc directly binds to ERK1, and complexes with ERK1,2 and MEK1. Intracellular RHAMM is required for sustained ERK1,2 activation and for nuclear localization of a subset of active ERK1,2. We hypothesized that this function results in expression of oncogenic proteins that support breast cancer cell aggression. Study purpose: determine the mechanism for RHAMMonc supported breast cancer aggression. Experimental procedure: we used microarray analysis in combination with real time PCR and ERK1,2 inhibition to identify genes that are upregulated in response to RHAMMonc overexpression. To determine whether nuclear RHAMMonc is required for neoplastic transformation we added a nuclear export signal to the RHAMMonc cDNA (RHAMMNES-onc). Mesenchymal cells overexpressing RHAMMonc or RHAMMNES-onc were injected into immune compromised mice. To test whether these cells could support in vivo growth of breast cancer cells, MCF7 cells were co-injected with RHAMMonc or RHAMMNES-onc expressing cells into the mammary gland fat pad. Tumor growth was quantified as tumor wet weight. Conclusion: Microarray analysis in combination with real time PCR identified altered expression of genes in all categories of the Hallmarks of Cancer in fibroblasts overexpressing RHAMMonc compared to parental cells. A subset of these were shown to be regulated by ERK1,2. Fusion of a nuclear export signal to RHAMMonc (RHAMMNES-onc) resulted in export of this protein from the nucleus, significant reduction of nuclear active ERK1,2 and modified expression of a subset of ERK1,2 regulated genes (e.g. LOX, MMP9, CDH11). Importantly, forced nuclear export of RHAMMonc suppressed its oncogenic effect on fibroblasts. Furthermore, proliferation of human MCF7 breast cancer cell xenografts was strongly stimulated by co-injection with RHAMMonc fibroblasts but this stimulatory effect was lost when MCF7 tumor cells were co-injected with RHAMMNES-onc. This study suggests that nuclear RHAMMonc:ERK 1,2 interactions control an oncogenic program in fibroblasts that support breast cancer aggression. Citation Format: Cornelia Toelg, Patrick Telmer, Sara Hamilton, James McCarthy, Eva Turley. Fibroblast RHAMM promotes breast cancer aggression by promoting expression of a subset of ERK1,2 target genes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1162. doi:10.1158/1538-7445.AM2014-1162