Reversal of Centrosome Amplification to Reduce Oncogenicity of Metastatic Uveal Melanoma

Centrosome amplification (CA), whereby cells have more than the normal number of centrosomes, is a common feature amongst aggressive cancers with a poor prognosis. CA drives oncogenic phenotypes such as increased invasiveness (via both cell-autonomous and non-cell-autonomous mechanisms) and chromosomal instability. In addition, CA can initiate tumourigenesis in flies and mice, and can drive advanced tumourigenic traits early on that promote disease progression. We hypothesise that reversal of CA may reverse CA-driven oncogenic phenotypes and therefore might be a new way to target aggressive cancers with a poor prognosis. This thesis presents the use of three patient-matched uveal melanoma (UM) cell lines as a new model to study CA that has developed in a patient setting. Mel270 cells were derived from a primary tumour and have negligible CA. OMM2.3 and OMM2.5 cells were derived from distinct liver metastases from the same patient and have high levels of CA. Primary UM can be well managed, however ~50% patients develop metastatic disease, for which there is no curative treatment and 1 year survival rates are ~50%, so there is a need to develop new therapies to target metastatic UM. Using RNA-Seq, genes that were differentially expressed in metastasis versus primary derived cells were selected for an siRNA screen to identify genes with a role in CA. Knockdown of either Aurora A or HSP90B1 induced a consistent reduction of CA in OMM2.3 cells. It is believed that Aurora A and HSP090B1 have independent effects on CA, as depleting either Aurora A or HSP90B1 using siRNAs did not affect the RNA or protein levels of the other. A new assay combining live imaging of migrating cells and immunofluorescence was developed and used, which we call FUCCI-CLIF (Fluorescent Ubiquitination based Cell Cycle Indicator - Correlative Live imaging and Immunofluorescence). FUCCI-CLIF provides insight into cell migration, cell cycle and CA status. Knockdown of Aurora A or HSP90B1 reduced migration as calculated by mean straight line speed. Further investigation indicated this was a non-cell-autonomous effect on cell migration. Ultimately, the work presented suggests that depleting or inhibiting proteins required for CA in a cancer specific setting reverses CA and concomitantly reduces oncogenic properties of aggressive metastatic UM cells, and is therefore a much needed new potential therapeutic approach against metastatic UM

Targeting centrosome amplification, an Achilles' heel of cancer

Due to cell-cycle dysregulation, many cancer cells contain more than the normal compliment of centrosomes, a state referred to as centrosome amplification (CA). CA can drive oncogenic phenotypes and indeed can cause cancer in flies and mammals. However, cells have to actively manage CA, often by centrosome clustering, in order to divide. Thus, CA is also an Achilles' Heel of cancer cells. In recent years, there have been many important studies identifying proteins required for the management of CA and it has been demonstrated that disruption of some of these proteins can cause cancer-specific inhibition of cell growth. For certain targets therapeutically relevant interventions are being investigated, for example, small molecule inhibitors, although none are yet in clinical trials. As the field is now poised to move towards clinically relevant interventions, it is opportune to summarise the key work in targeting CA thus far, with particular emphasis on recent developments where small molecule or other strategies have been proposed. We also highlight the relatively unexplored paradigm of reversing CA, and thus its oncogenic effects, for therapeutic gain

Aggressive uveal melanoma displays a high degree of centrosome amplification, opening the door to therapeutic intervention

Abstract: Uveal melanoma (UM) is the most common intraocular cancer in adults. Whilst treatment of primary UM (PUM) is often successful, around 50% of patients develop metastatic disease with poor outcomes, linked to chromosome 3 loss (monosomy 3, M3). Advances in understanding UM cell biology may indicate new therapeutic options. We report that UM exhibits centrosome abnormalities, which in other cancers are associated with increased invasiveness and worse prognosis, but also represent a potential Achilles' heel for cancer‐specific therapeutics. Analysis of 75 PUM patient samples revealed both higher centrosome numbers and an increase in centrosomes with enlarged pericentriolar matrix (PCM) compared to surrounding normal tissue, both indicative of centrosome amplification. The PCM phenotype was significantly associated with M3 (t‐test, p <0.01). Centrosomes naturally enlarge as cells approach mitosis; however, whilst UM with higher mitotic scores had enlarged PCM regardless of genetic status, the PCM phenotype remained significantly associated with M3 in UM with low mitotic scores (ANOVA, p = 0.021) suggesting that this is independent of proliferation. Phenotypic analysis of patient‐derived cultures and established UM lines revealed comparable levels of centrosome amplification in PUM cells to archetypal triple‐negative breast cancer cell lines, whilst metastatic UM (MUM) cell lines had even higher levels. Importantly, many UM cells also exhibit centrosome clustering, a common strategy employed by other cancer cells with centrosome amplification to survive cell division. As UM samples with M3 display centrosome abnormalities indicative of amplification, this phenotype may contribute to the development of MUM, suggesting that centrosome de‐clustering drugs may provide a novel therapeutic approach