Christopher A. Maxwell

Cell-surface and mitotic-spindle RHAMM: moonlighting or dual oncogenic functions?

Tumor cells use a wide variety of post-translational mechanisms to modify the functional repertoire of their transcriptome. One emerging but still understudied mechanism involves the export of cytoplasmic proteins that then partner with cell-surface receptors and modify both the surface-display kinetics and signaling properties of these receptors. Recent investigations demonstrate moonlighting roles for the proteins epimorphin, FGF1, FGF2, PLK1 and Ku80, to name a few, during oncogenesis and inflammation. Here, we review the molecular mechanisms of unconventional cytoplasmic-protein export by focusing on the mitotic-spindle/hyaluronan-binding protein RHAMM, which is hyper-expressed in many human tumors. Intracellular RHAMM associates with BRCA1 and BARD1; this association attenuates the mitotic-spindle-promoting activity of RHAMM that might contribute to tumor progression by promoting genomic instability. Extracellular RHAMM-CD44 partnering sustains CD44 surface display and enhances CD44-mediated signaling through ERK1 and ERK2 (ERK1/2); it might also contribute to tumor progression by enhancing and/or activating the latent tumor-promoting properties of CD44. The unconventional export of proteins such as RHAMM is a novel process that modifies the roles of tumor suppressors and promoters, such as BRCA1 and CD44, and might provide new targets for therapeutic intervention.

Hyaluronan and Hyaluronan Synthases: Potential Therapeutic Targets in Cancer

Current models of oncogenesis describe cancer as a progression of genetic mutations in a tumor cell mass. However, tumors are more than a clonal expansion of malignant cells. Tumors are heterogeneous, with a complex 3D structure, analogous to organs comprised of different tissues. In a tumor mass, the component cell types interact with each other and with their microenvironment by exchanging information through cell-cell interactions and/or through interactions with the extracellular matrix (ECM). These synergetic interactions facilitate tumor progression. Furthermore, tumor invasion and metastatic development are accomplished through the breakdown of ECM. Disruption of ECM promotes abnormal inter- and/or intra- cellular signaling, leading to dysregulation of cell proliferation, growth and cytoskeleton reorganization. The disruption of the ECM in turn promotes the overproduction of growth factors, which induce elevated epithelial cell proliferation and other abnormalities including carcinogenesis. In this review we will demonstrate that hyaluronan (HA), a core component of ECM, contributes to certain types of cancer development. Additional to extracellular HA, intracellular and nuclear forms of HA have been detected. Intracellular HA is involved in cell signaling, whereas nuclear HA could promote chromatin condensation and thus facilitate mitosis. HA molecules are synthesized by hyaluronan synthases (HASs)-HAS1, HAS2 and HAS3 enzymes. Dysregulation of HAS genes results in abnormal production of HA and promotion of abnormal biological processes such as transformation and metastasis. The function of HASs appears to be cell and tissue specific. HAS1 maintains a low, basal level of HA. HAS2 is involved in embryonic and cardiac cushion morphogenesis and subsequent development through cell migration and invasion. HAS2 stimulates cell proliferation and angiogenesis. HAS3 appears to favor the malignant phenotype in many types of malignancies. However, the exact function of HAS isoenzymes and their role in cell signaling remains to be elucidated. A better understanding of HA and HASs may facilitate the design of novel therapeutic strategies to counter presumptive cancer-promoting effects of microenvironmental components.

Genomic instability in human cancer: Molecular insights and opportunities for therapeutic attack and prevention through diet and nutrition

Genomic instability can initiate cancer, augment progression, and influence the overall prognosis of the affected patient. Genomic instability arises from many different pathways, such as telomere damage, centrosome amplification, epigenetic modifications, and DNA damage from endogenous and exogenous sources, and can be perpetuating, or limiting, through the induction of mutations or aneuploidy, both enabling and catastrophic. Many cancer treatments induce DNA damage to impair cell division on a global scale but it is accepted that personalized treatments, those that are tailored to the particular patient and type of cancer, must also be developed. In this review, we detail the mechanisms from which genomic instability arises and can lead to cancer, as well as treatments and measures that prevent genomic instability or take advantage of the cellular defects caused by genomic instability. In particular, we identify and discuss five priority targets against genomic instability: (1) prevention of DNA damage; (2) enhancement of DNA repair; (3) targeting deficient DNA repair; (4) impairing centrosome clustering; and, (5) inhibition of telomerase activity. Moreover, we highlight vitamin D and B, selenium, carotenoids, PARP inhibitors, resveratrol, and isothiocyanates as priority approaches against genomic instability. The prioritized target sites and approaches were cross validated to identify potential synergistic effects on a number of important areas of cancer biology.

The selective Aurora B kinase inhibitor AZD1152 is a potential new treatment for multiple myeloma

Aurora kinases are potential targets for cancer therapy. Previous studies have validated Aurora kinase A as a therapeutic target in multiple myeloma (MM), and have demonstrated in vitro anti‐myeloma effects of small molecule Aurora kinase inhibitors that inhibit both Aurora A and B. This study demonstrated that Aurora B kinase was strongly expressed in myeloma cell lines and primary plasma cells. The selective Aurora B inhibitor AZD1152‐induced apoptotic death in myeloma cell lines at nanomolar concentrations, with a cell cycle phenotype consistent with that reported previously for Aurora B inhibition. In some cases, AZD1152 in combination with dexamethasone showed increased anti‐myeloma activity compared with the use of either agent alone. AZD1152 was active against sorted CD138+ BM plasma cells from myeloma patients but also, as expected, was toxic to CD138− marrow cells from the same patients. In a murine myeloma xenograft model, AZD1152‐inhibited tumour growth at well‐tolerated doses and induced cell death in established tumours, with associated mild, transient leucopenia. AZD1152 shows promise in these preclinical studies as a novel treatment for MM.

Targeted and Nontargeted Effects of Ionizing Radiation That Impact Genomic Instability

Radiation-induced genomic instability, in which the progeny of irradiated cells display a high frequency of nonclonal genomic damage, occurs at a frequency inconsistent with mutation. We investigated the mechanism of this nontargeted effect in human mammary epithelial cells (HMEC) exposed to low doses of radiation. We identified a centrosome-associated expression signature in irradiated HMEC and show here that centrosome deregulation occurs in the first cell cycle after irradiation, is dose dependent, and that viable daughters of these cells are genomically unstable as evidenced by spontaneous DNA damage, tetraploidy, and aneuploidy. Clonal analysis of genomic instability showed a threshold of >10 cGy. Treatment with transforming growth factor beta1 (TGFbeta), which is implicated in regulation of genomic stability and is activated by radiation, reduced both the centrosome expression signature and centrosome aberrations in irradiated HMEC. Furthermore, TGFbeta inhibition significantly increased centrosome aberration frequency, tetraploidy, and aneuploidy in nonirradiated HMEC. Rather than preventing radiation-induced or spontaneous centrosome aberrations, TGFbeta selectively deleted unstable cells via p53-dependent apoptosis. Together, these studies show that radiation deregulates centrosome stability, which underlies genomic instability in normal human epithelial cells, and that this can be opposed by radiation-induced TGFbeta signaling.

Geometric approach to segmentation and protein localization in cell culture assays

Cell‐based fluorescence imaging assays are heterogeneous and require the collection of a large number of images for detailed quantitative analysis. Complexities arise as a result of variation in spatial nonuniformity, shape, overlapping compartments and scale (size). A new technique and methodology has been developed and tested for delineating subcellular morphology and partitioning overlapping compartments at multiple scales. This system is packaged as an integrated software platform for quantifying images that are obtained through fluorescence microscopy. Proposed methods are model based, leveraging geometric shape properties of subcellular compartments and corresponding protein localization. From the morphological perspective, convexity constraint is imposed to delineate and partition nuclear compartments. From the protein localization perspective, radial symmetry is imposed to localize punctate protein events at submicron resolution. Convexity constraint is imposed against boundary information, which are extracted through a combination of zero‐crossing and gradient operator. If the convexity constraint fails for the boundary then positive curvature maxima are localized along the contour and the entire blob is partitioned into disjointed convex objects representing individual nuclear compartment, by enforcing geometric constraints. Nuclear compartments provide the context for protein localization, which may be diffuse or punctate. Punctate signal are localized through iterative voting and radial symmetries for improved reliability and robustness. The technique has been tested against 196 images that were generated to study centrosome abnormalities. Corresponding computed representations are compared against manual counts for validation.

YB-1 evokes susceptibility to cancer through cytokinesis failure, mitotic dysfunction, and HER2 amplification

Y-box binding protein-1 (YB-1) expression in the mammary gland promotes breast carcinoma that demonstrates a high degree of genomic instability. In the present study, we developed a model of pre-malignancy to characterize the role of this gene during breast cancer initiation and early progression. Antibody microarray technology was used to ascertain global changes in signal transduction following the conditional expression of YB-1 in human mammary epithelial cells (HMEC). Cell cycle-associated proteins were frequently altered with the most dramatic being LIM kinase 1/2 (LIMK1/2). Consequently, the misexpression of LIMK1/2 was associated with cytokinesis failure that acted as a precursor to centrosome amplification. Detailed investigation revealed that YB-1 localized to the centrosome in a phosphorylation-dependent manner, where it complexed with pericentrin and γ-tubulin. This was found to be essential in maintaining the structural integrity and microtubule nucleation capacity of the organelle. Prolonged exposure to YB-1 led to rampant acceleration toward tumorigenesis, with the majority of cells acquiring numerical and structural chromosomal abnormalities. Slippage through the G1/S checkpoint due to overexpression of cyclin E promoted continued proliferation of these genomically compromised cells. As malignancy further progressed, we identified a subset of cells harboring HER2 amplification. Our results recognize YB-1 as a cancer susceptibility gene, with the capacity to prime cells for tumorigenesis.

TACC3-TSC2 maintains nuclear envelope structure and controls cell division

Studies of the role of tuberous sclerosis complex (TSC) proteins (TSC1/TSC2) in pathology have focused mainly on their capacity to regulate translation and cell growth, but their relationship with alterations of cellular structures and the cell cycle is not yet fully understood. The transforming acidic coiled-coil (TACC) domain-containing proteins are central players in structures and processes connected to the centrosome. Here, TACC3 interactome mapping identified TSC2 and 15 other physical interactors, including the evolutionary conserved interactions with ch-TOG/CKAP5 and FAM161B. TACC3 and TSC2 co-localize and co-purify with components of the nuclear envelope, and their deficiency causes morphological alterations of this structure. During cell division, TACC3 is necessary for the proper localization of phospho-Ser939 TSC2 at spindle poles and cytokinetic bridges. Accordingly, abscission alterations and increased frequency of binucleated cells were observed in Tacc3- and Tsc2-deficient cells relative to controls. In regulating cell division, TSC2 acts epistatically to TACC3 and, in addition to canonical TSC/mTOR signaling and cytokinetic associations, converges to the early mitotic checkpoint mediated by CHFR, consistently with nuclear envelope associations. Our findings link TACC3 to novel structural and cell division functions of TSC2, which may provide additional explanations for the clinical and pathological manifestations of lymphangioleiomyomatosis (LAM) disease and TSC syndrome, including the greater clinical severity of TSC2 mutations compared to TSC1 mutations.

Genetic and genomic analysis modeling of germline c-MYC overexpression and cancer susceptibility

BackgroundGermline genetic variation is associated with the differential expression of many human genes. The phenotypic effects of this type of variation may be important when considering susceptibility to common genetic diseases. Three regions at 8q24 have recently been identified to independently confer risk of prostate cancer. Variation at 8q24 has also recently been associated with risk of breast and colorectal cancer. However, none of the risk variants map at or relatively close to known genes, with c-MYC mapping a few hundred kilobases distally.ResultsThis study identifies cis-regulators of germline c-MYC expression in immortalized lymphocytes of HapMap individuals. Quantitative analysis of c-MYC expression in normal prostate tissues suggests an association between overexpression and variants in Region 1 of prostate cancer risk. Somatic c-MYC overexpression correlates with prostate cancer progression and more aggressive tumor forms, which was also a pathological variable associated with Region 1. Expression profiling analysis and modeling of transcriptional regulatory networks predicts a functional association between MYC and the prostate tumor suppressor KLF6. Analysis of MYC/Myc-driven cell transformation and tumorigenesis substantiates a model in which MYC overexpression promotes transformation by down-regulating KLF6. In this model, a feedback loop through E-cadherin down-regulation causes further transactivation of c-MYC.ConclusionThis study proposes that variation at putative 8q24 cis-regulator(s) of transcription can significantly alter germline c-MYC expression levels and, thus, contribute to prostate cancer susceptibility by down-regulating the prostate tumor suppressor KLF6 gene.

Biological convergence of cancer signatures

Gene expression profiling has identified cancer prognostic and predictive signatures with superior performance to conventional histopathological or clinical parameters. Consequently, signatures are being incorporated into clinical practice and will soon influence everyday decisions in oncology. However, the slight overlap in the gene identity between signatures for the same cancer type or condition raises questions about their biological and clinical implications. To clarify these issues, better understanding of the molecular properties and possible interactions underlying apparently dissimilar signatures is needed. Here, we evaluated whether the signatures of 24 independent studies are related at the genome, transcriptome or proteome levels. Significant associations were consistently observed across these molecular layers, which suggest the existence of a common cancer cell phenotype. Convergence on cell proliferation and death supports the pivotal involvement of these processes in prognosis, metastasis and treatment response. In addition, functional and molecular associations were identified with the immune response in different cancer types and conditions that complement the contribution of cell proliferation and death. Examination of additional, independent, cancer datasets corroborated our observations. This study proposes a comprehensive strategy for interpreting cancer signatures that reveals common design principles and systems-level properties.