Helen Chen

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.

Multifunctional Proteins Bridge Mitosis with Motility and Cancer with Inflammation and Arthritis

While most secreted proteins contain defined signal peptides that direct their extracellular transport through the ER-Golgi pathway, nonclassical transport of leaderless peptides/proteins was first described 20 years ago and the mechanisms responsible for unconventional export of such proteins have been thoroughly reviewed. In addition to directed nonclassical secretion, a number of leaderless secreted proteins have been classified as damage-associated molecular-pattern (DAMP) molecules, which are nuclear or cytoplasmic proteins that, under necrotic or apoptotic conditions, are released outside the cell and function as proinflammatory signals. A strong association between persistent release of DAMPs, chronic inflammation, and the hypoxic tumor microenvironment has been proposed. Thus, protein localization and function can change fundamentally from intracellular to extracellular compartments, often under conditions of inflammation, cancer, and arthritis. If we are truly to understand, model, and treat such biological states, it will be important to investigate these multifunctional proteins and their contribution to degenerative diseases. Here, we will focus our discussion on intracellular proteins, both cytoplasmic and nuclear, that play critical extracellular roles. In particular, the multifunctional nature of HMMR/RHAMM and survivin will be highlighted and compared, as these molecules are the subject of extensive biological and therapeutic investigations within hematology and oncology fields. For these and other genes/proteins, we will highlight points of structural and functional intersection during cellular division and differentiation, as well as states associated with cancer, such as tumor-initiation and epithelial-to-mesenchymal transition (EMT). Finally, we will discuss the potential targeting of these proteins for improved therapeutic outcomes within these degenerative disorders. Our goal is to highlight a number of commonalities among these multifunctional proteins for better understanding of their putative roles in tumor initiation, inflammation, arthritis, and cancer.

Spatial regulation of Aurora A activity during mitotic spindle assembly requires RHAMM to correctly localize TPX2

Construction of a mitotic spindle requires biochemical pathways to assemble spindle microtubules and structural proteins to organize these microtubules into a bipolar array. Through a complex with dynein, the receptor for hyaluronan-mediated motility (RHAMM) cross-links mitotic microtubules to provide structural support, maintain spindle integrity, and correctly orient the mitotic spindle. Here, we locate RHAMM to sites of microtubule assembly at centrosomes and non-centrosome sites near kinetochores and demonstrate that RHAMM is required for the activation of Aurora kinase A. Silencing of RHAMM delays the kinetics of spindle assembly, mislocalizes targeting protein for XKlp2 (TPX2), and attenuates the localized activation of Aurora kinase A with a consequent reduction in mitotic spindle length. The RHAMM–TPX2 complex requires a C-terminal basic leucine zipper in RHAMM and a domain that includes the nuclear localization signal in TPX2. Together, our findings identify RHAMM as a critical regulator for Aurora kinase A signaling and suggest that RHAMM ensures bipolar spindle assembly and mitotic progression through the integration of biochemical and structural pathways.

HMMR acts in the PLK1-dependent spindle positioning pathway and supports neural development

Oriented cell division is one mechanism progenitor cells use during development and to maintain tissue homeostasis. Common to most cell types is the asymmetric establishment and regulation of cortical NuMA-dynein complexes that position the mitotic spindle. Here, we discover that HMMR acts at centrosomes in a PLK1-dependent pathway that locates active Ran and modulates the cortical localization of NuMA-dynein complexes to correct mispositioned spindles. This pathway was discovered through the creation and analysis of Hmmr-knockout mice, which suffer neonatal lethality with defective neural development and pleiotropic phenotypes in multiple tissues. HMMR over-expression in immortalized cancer cells induces phenotypes consistent with an increase in active Ran including defects in spindle orientation. These data identify an essential role for HMMR in the PLK1-dependent regulatory pathway that orients progenitor cell division and supports neural development.

Cell Cycle–Dependent Tumor Engraftment and Migration Are Enabled by Aurora-A

Cell-cycle progression and the acquisition of a migratory phenotype are hallmarks of human carcinoma cells that are perceived as independent processes but may be interconnected by molecular pathways that control microtubule nucleation at centrosomes. Here, cell-cycle progression dramatically impacts the engraftment kinetics of 4T1-luciferase2 breast cancer cells in immunocompetent BALB/c or immunocompromised NOD-SCID gamma (NSG) mice. Multiparameter imaging of wound closure assays was used to track cell-cycle progression, cell migration, and associated phenotypes in epithelial cells or carcinoma cells expressing a fluorescence ubiquitin cell-cycle indicator. Cell migration occurred with an elevated velocity and directionality during the S–G2-phase of the cell cycle, and cells in this phase possess front-polarized centrosomes with augmented microtubule nucleation capacity. Inhibition of Aurora kinase-A (AURKA/Aurora-A) dampens these phenotypes without altering cell-cycle progression. During G2-phase, the level of phosphorylated Aurora-A at centrosomes is reduced in hyaluronan-mediated motility receptor (HMMR)-silenced cells as is the nuclear transport of TPX2, an Aurora-A–activating protein. TPX2 nuclear transport depends upon HMMR-T703, which releases TPX2 from a complex with importin-α (KPNA2) at the nuclear envelope. Finally, the abundance of phosphorylated HMMR-T703, a substrate for Aurora-A, predicts breast cancer–specific survival and relapse-free survival in patients with estrogen receptor (ER)–negative (n = 941), triple-negative (TNBC) phenotype (n = 538), or basal-like subtype (n = 293) breast cancers, but not in those patients with ER-positive breast cancer (n = 2,218). Together, these data demonstrate an Aurora-A/TPX2/HMMR molecular axis that intersects cell-cycle progression and cell migration. Implications: Tumor cell engraftment, migration, and cell-cycle progression share common regulation of the microtubule cytoskeleton through the Aurora-A/TPX2/HMMR axis, which has the potential to influence the survival of patients with ER-negative breast tumors. Mol Cancer Res; 16(1); 16–31. ©2017 AACR.

The non-motor adaptor HMMR dampens Eg5-mediated forces to preserve the kinetics and integrity of chromosome segregation

The nonmotor adaptor protein HMMR maintains the kinetics and integrity of chromosome segregation by promoting TPX2-Eg5 complexes that dampen Eg5-mediated forces and support K-fiber stability, kinetochore–microtubule attachments, and inter-kinetochore tension. HMMR is needed to prevent the generation of aneuploid progeny cells.

The Generation, Detection, and Prevention of Genomic Instability During Cancer Progression and Metastasis

Genome stability is tightly regulated through the cell cycle. Aberrations in genome structure and sequence are a hallmark of malignancy and these changes can allow abnormal cells to escape the regulatory mechanisms that would otherwise direct these cells into apoptosis or senescence. When genome instability occurs, it can happen as large or small structural changes in the genome, changes in gene expression, or even changes at the epigenetic level. There are many environmental factors that can induce DNA damage and strain the machinery that is responsible for maintaining genome stability. In some cases, such as UV light or chemical carcinogens, it is possible to avoid these factors and thus reduce the risk of cancer. But, in other instances, hereditary mutations impair the function of genes and their products, which normally protect the stability of the genome. While genomic instability offers selective advantages to the tumor, the tumor-specific loss of these pathways may provide therapeutic opportunities, which could be personalized through knowledge of the specific types of genomic instability that characterize an individual’s tumor.

Abstract LB-214: Common genomic alterations in malignant peripheral nerve sheath tumors augment Aurora A activity and sensitize tumors to aurora kinase inhibitors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnMalignant peripheral nerve sheath tumours (MPNST) are rare, hereditary, cancers associated with mutations in the neurofibromin 1 gene [1][1]. MPNSTs are often resistant to chemotherapies and have high rates of disease recurrence, highlighting the lack of effective treatment options for this cancer. Aurora kinase A inhibitors (AKIs) have shown promise against MPNST cell lines [2][2]. We expanded this study by testing AKI in human MPNST xenotransplant mice models. Treatment resulted in stabilized disease with tumor cells undergoing senescence and endoreduplication.nnAurora kinase A (AURKA) is an emerging target in cancer, however, targeted therapies can often fail in the clinic due to insufficient knowledge about factors that determine tumor response. Therefore, we utilized three MPNST cell lines and profiled them for the expression and activity of AURKA as well as their responses to AKIs. The most proliferative lines, S462 and 2884, express equivalent levels of AURKA, however, S462 cells were more sensitive to kinase inhibition. Both cell lines experienced apoptosis, senescence and endoreduplication in response to AKI treatment.nnAURKA activity is regulated by a co-activator, the Targeting Protein for XKlp2 (TPX2) and a molecular brake, the Receptor for Hyaluronan Mediated Motility (RHAMM)[3][3]. Interestingly, published analysis of copy number variation has identified hemizygous loss of the RHAMM gene in half of the examined high-grade MPNST, but not in benign or low grade tumors [4][4]. We proposed that MPNSTs with RHAMM deletions are oncogene addicted to AURKA activity and are therefore, particularly susceptible to AKI. We profiled our MPNST lines for RHAMM and TPX2 expression and found that S462 cells express significantly more TPX2 and less RHAMM compared to 2884 cells. Furthermore, S462 cells had increased kinase. To determine whether levels of these molecular regulators could affect AKI efficacy we depleted RHAMM and TPX2 in 2884 and S462 cells respectively. While cells with reduced TPX2 have unchanged responses to AKIs, RHAMM depleted cells have a 2 fold reduction in IC-50s.nnWe also looked at the effect of AKI against a population of MPNST tumor-initiating cells (TICs) from the S462 line. Compared to adherent cells, S462 TICs have elevated AURKA activity and their ability to self-renew in vitro is arrested by AKI. Indeed, the altered levels of kinase activity in the RHAMM and TPX2 depletion lines correlated with their ability to form and maintain sphere culture. In addition, we find that AKI treated S462 TICs differentiated into terminal neurons.nnAll in all, these data indicate AURKA as a rational therapy for aggressive MPNSTs with RHAMM serving as a biomarker for AKI efficacy.nnCitation Format: Pooja Mohan, Joan Castellsague, Jihong Jiang, Kristi Allen, Helen Chen, Oksana Nemirovsky, Melanie Spyra, Kaiji Hu, Lan Kluwe, Miguel Pujana, Alberto Villanueva, Victor Mautner, Sandra Dunn, Jonathan Keats, Conxi Lazaro, Christopher Maxwell. Common genomic alterations in malignant peripheral nerve sheath tumors augment Aurora A activity and sensitize tumors to aurora kinase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2013-LB-214nn [1]: #ref-1n [2]: #ref-2n [3]: #ref-3n [4]: #ref-4

Abstract LB-124: RHAMM acts near chromosomes to promote mitotic spindle assembly by regulating the location and abundance of TPX2 and the downstream activation of Aurora A.

Enhanced cell proliferation is a hallmark of cancer. Thus, molecular pathways underlying mitosis are important targets for therapy. To form a bipolar mitotic spindle, the dividing cell must assemble microtubules at centrosomes [spindle poles] as well as non-centrosome sites [kinetochores]. The spindle then captures and aligns the duplicated chromosomes. Correct attachment satisfies the spindle assembly checkpoint (SAC) and allows for mitotic progression. Aurora kinase A, when bound by its activator, TPX2, is a key regulator of spindle assembly. Upon activation, the kinase phosphorylates substrates to target them to the centrosomes, and protects them from proteolytic degradation. Since it is necessary for optimal Aurora A function, TPX2 availability is tightly controlled. The receptor for hyaluronan-mediated motility (RHAMM) is a microtubule associated protein that interacts with TPX2 and participates in spindle assembly. However, it is unclear whether or how the RHAMM-TPX2 complex modulates Aurora A activity. We hypothesize that RHAMM interacts with TPX2 to determine TPX2 location and abundance, which facilitates Aurora A activity and spindle assembly. We have followed the location and interactions of RHAMM through mitosis, as well as the consequences for its loss of function. We tracked the location of RHAMM and GFP-RHAMM and found it at the kinetochores. Then, we colocalized RHAMM with key kinetochore proteins, BUBR1 and NDC80, during microtubule nucleation and confirmed physical associations by IP. For insights into the function of RHAMM, we followed mitotic cells in real-time. In HeLa cells treated with siRNA targeting RHAMM, we found a significant increase in aberrant spindle morphology and mitotic failure, and we were able to rescue these phenotypes by reintroducing GFP-RHAMM. We also measured the duration of spindle assembly, chromosome congression and cytokinesis. RHAMM depleted mitotic cells present two distinct phenotypes, and these cells either fail spindle assembly or construct phenotypically “normal” mitotic spindles that do not satisfy the SAC. Thus, the protein9s location, interactions, and the consequences of its depletion all point to a critical role at the kinetochore. Previous reports suggest RHAMM participates in spindle assembly by interacting with TPX2, however, the exact mechanism remains unknown. We find that RHAMM depletion decreases TPX2 protein abundance during early mitosis, and impairs TPX2 transit to the spindle poles, which results in reduced Aurora A protein abundance and activity. Together, these results suggest that RHAMM influences Aurora A actions by regulating the stability and location of TPX2. Further insights into RHAMM associated pathways may lead to the discovery of new therapeutic targets and optimize the usage of promising drugs such as Aurora A inhibitors. Citation Format: Helen Chen, Chinten Lim, Chris Maxwell. RHAMM acts near chromosomes to promote mitotic spindle assembly by regulating the location and abundance of TPX2 and the downstream activation of Aurora A. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-124. doi:10.1158/1538-7445.AM2013-LB-124