Genetic analysis identifies the HMMR gene as a modifier of the breast cancer risk associated with BRCA1 gene mutation, while cell biological analysis of the protein product suggests a function in regulating development of the mammary gland.

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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-G-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 G-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 ( = 941), triple-negative (TNBC) phenotype ( = 538), or basal-like subtype ( = 293) breast cancers, but not in those patients with ER-positive breast cancer ( = 2,218). Together, these data demonstrate an Aurora-A/TPX2/HMMR molecular axis that intersects cell-cycle progression and cell migration. 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. .

Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary cancers associated with neurofibromatosis type I. MPNSTs lack effective treatment options as they often resist chemotherapies and have high rates of disease recurrence. Aurora kinase A (AURKA) is an emerging target in cancer and an aurora kinase inhibitor (AKI), termed MLN8237, shows promise against MPNST cell lines in vitro and in vivo. Here, we test MLN8237 against two primary human MPNST grown in vivo as xenotransplants and find that treatment results in tumour cells exiting the cell cycle and undergoing endoreduplication, which cumulates in stabilized disease. Targeted therapies can often fail in the clinic due to insufficient knowledge about factors that determine tumour susceptibilities, so we turned to three MPNST cell-lines to further study and modulate the cellular responses to AKI. We find that the sensitivity of cell-lines with amplification of AURKA depends upon the activity of the kinase, which correlates with the expression of the regulatory gene products TPX2 and HMMR/RHAMM. Silencing of HMMR/RHAMM, but not TPX2, augments AURKA activity and sensitizes MPNST cells to AKI. Furthermore, we find that AURKA activity is critical to the propagation and self-renewal of sphere-enriched MPNST cancer stem-like cells. AKI treatment significantly reduces the formation of spheroids, attenuates the self-renewal of spheroid forming cells, and promotes their differentiation. Moreover, silencing of HMMR/RHAMM is sufficient to endow MPNST cells with an ability to form and maintain sphere culture. Collectively, our data indicate that AURKA is a rationale therapeutic target for MPNST and tumour cell responses to AKI, which include differentiation, are modulated by the abundance of HMMR/RHAMM.

Receptor for hyaluronan mediated motility (RHAMM, encoded by HMMR) may be a cell-surface receptor for hyaluronan that regulates embryonic stem cell pluripotency and differentiation, however, a precise mechanism for its action is not known. We examined murine embryonic stem cells with and without hemizygous genomic mutation of Hmmr/RHAMM, but we were not able to find RHAMM on the cell-surface. Rather, RHAMM localized to the microtubule cytoskeleton and along mitotic spindles. Genomic loss of Hmmr/RHAMM did not alter cell cycle progression but augmented differentiation and attenuated pluripotency in murine embryonic stem cells. Through a candidate screen of small-molecule kinase inhibitors, we identified ERK1/2 and aurora kinase A as barrier kinases whose inhibition was sufficient to rescue pluripotency in RHAMM+/- murine embryonic stem cells. Thus, RHAMM is not found on the cell-surface of embryonic stem cells, but it is required to maintain pluripotency and its dominant mechanism of action is through the modulation of signal transduction pathways at microtubules.

Most patients with tuberous sclerosis complex (TSC) develop cortical tubers that cause severe neurological disabilities. It has been suggested that defects in neuronal differentiation and/or migration underlie the appearance of tubers. However, the precise molecular alterations remain largely unknown. Here, by combining cytological and immunohistochemical analyses of tubers from nine TSC patients (four of them diagnosed with TSC2 germline mutations), we show that alteration of microtubule biology through ROCK2 signalling contributes to TSC neuropathology. All tubers showed a larger number of binucleated neurons than expected relative to control cortex. An excess of normal and altered cytokinetic figures was also commonly observed. Analysis of centrosomal markers suggested increased microtubule nucleation capacity, which was supported by the analysis of an expression dataset from cortical tubers and control cortex, and subsequently linked to under-expression of Rho-associated coiled-coil containing kinase 2 (ROCK2). Thus, augmented microtubule nucleation capacity was observed in mouse embryonic fibroblasts and human fibroblasts deficient in the Tsc2/TSC2 gene product, tuberin. Consistent with ROCK2 under-expression, microtubule acetylation was found to be increased with tuberin deficiency; this alteration was abrogated by rapamycin treatment and mimicked by HDAC6 inhibition. Together, the results of this study support the hypothesis that loss of TSC2 expression can alter microtubule organization and dynamics, which, in turn, deregulate cell division and potentially impair neuronal differentiation.

Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary, cancers associated with mutations in the neurofibromin 1 gene 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. 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. Aurora 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. AURKA 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. 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. 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. We 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. All in all, these data indicate AURKA as a rational therapy for aggressive MPNSTs with RHAMM serving as a biomarker for AKI efficacy. Citation 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-214

PROBLEM- Metastasis accounts for 90% of deaths from carcinomas. BACKGROUND- To initiate migration, epithelial cells must front-rear polarize and reorganize the microtubule cytoskeleton directed toward the lamillipodia or leading edge. During mitosis, centrosome maturation, duplication, spindle pole assembly and chromosome segregation require Aurora kinase A (AURKA), which is optimally active when complexed with TPX2. Accumulating evidences identify non-mitotic functions for AURKA, such as cell migration and ciliary reorganization. Overexpression of AURKA leads to tumorigenesis in breast cancer. Mechanisms that activate non-mitotic AURKA and promote cell migration as well as metastasis are yet to be uncovered. RESULTS- In a large breast cancer tissue microarray (n= 3,175), the abundance of phosphorylated RHAMM (a substrate of AURKA) predicted overall and relapse-free survival in ER-negative, basal-subtype, and triple negative breast tumors. We studied the AURKA-TPX2-RHAMM signaling axis in mammary (MCF10A and nMUMG-FUCCI) and HeLa cells. In scratch wound assays, migratory cells were found to have front-polarized centrosomes, which correlated with G2 cell cycle phase, phosphorylated AURKA and elevated microtubule nucleation at centrosomes. Small molecule inhibition of Aurora kinase activity impaired centrosome polarity and retarded the kinetics of cell migration. In parallel, silencing RHAMM altered TPX2 location as indicated by immunofluorescence and immuno-EM, which located TPX2 to the nuclear envelope and nuclear pore basket. Nuclear import of recombinant TPX2 was attenuated in RHAMM silenced cells and nuclear envelope accumulation was elevated. CONCLUSION- The AURKA-TPX2-RHAMM axis regulates centrosome polarity, microtubule nucleation and directional cell migration while phosphorylated RHAMM predicts survival in aggressive forms of breast cancer. Citation Format: Tony Lok Heng Chu, Lixin Zhou, Jennifer Won, Pooja Mohan, Oksana Nemirovsky, Abbas Fotovati, Torsten Nielsen, Nelly Pante, Christopher Maxwell. Cell cycle-dependent front polarized cell migration requires Aurora kinase A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1876. doi:10.1158/1538-7445.AM2017-1876