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Mitotis-induced entosis and its role in cancer

Final Report Summary - ENTOSIS2013 (Mitotis-induced entosis and its role in cancer)

Mitosis-induced entosis and its role in cancer

Entosis is an intriguing form of cellular cannibalism that is commonly observed in human tumours. Entosis has previously been shown to be triggered when cells become detached from their underlying extracellular matrix, as often occurs during cancer. In this project, we have now discovered that cell proliferation (mitosis) can also act as a trigger for cell cannibalism through entosis, and have elucidated key molecular and cellular mechanisms underlying this phenomenon. Given that uncontrolled cell proliferation is a hallmark of cancer, this novel link with cell cannibalism is particularly striking. Related to this, we have identified a clear correlation between frequency of mitosis and cell cannibalism in human breast cancers, and have identified an unexpected relationship between some commonly used chemotherapeutics and entosis. These studies have provided important new insights into the emerging field of cell cannibalism in cancer, and open up important new lines of research to develop through future work.

The following summary describes the work carried out to achieve the project’s specific objectives, the main results and conclusions and the wider impact of this study:

Aims 1 and 2: Molecular and Biophysical Mechanisms of Mitosis-Induced Entosis

To investigate the molecular mechanisms underlying mitotic entosis, the following experimental approaches were employed:

1) Candidate gene manipulation: Using RNAi-based approaches, specific genes were depleted and their effects on mitotic entosis assessed. Cdc42, a Rho-family small GTPase with a major role in epithelial cell biology, was a primary focus, based on preliminary data. A series of candidate Cdc42 effectors (e.g. MRCK, Borg, MYPT1) were also investigated, as well as b1-integrin, a protein that controls cell binding to, and communication with, the underlying extracellular matrix, and can be regulated by Cdc42. Finally, a related small GTPase, Rap1, was also investigated using a dominant negative based approach; Rap1 is known to specifically regulate cell-matrix adhesion during mitosis. Following all of these manipulations,both mitosis and entosis were analysed and measured using a variety of microscopic techniques, including timelapse, confocal and 3D-Correlative Light Electron Microscopy (3D-CLEM).

2) Fluorescence Resonance Energy Transfer (FRET): RhoA is another small GTPase that can regulate entosis. To explore the relationship between Cdc42 and RhoA during mitosis, an established FRET probe was employed to measure spatiotemporal RhoA activation throughout the cell cycle and in the presence and absence of Cdc42 activity.

3) Pharmacological inhibitors: A series of inhibitors were employed to interrogate components of the Rho signaling pathway during mitotic entosis: C3 transferase (a RhoA inhibitor), Y27632 (a ROCK inhibitor) and blebbistatin (a myosin II inhibitor). A panel of small molecules were used to arrest cells at different points in the cell cycle: RO-3306 (Cdk1 inhibitor, G2/M arrest), Paclitaxel/taxol, nocodazole and STLC (prometaphase arrest).

4) Biophysics: We are engaged in an ongoing collaboration to directly measure biophysical cell properties using real-time deformability cytometry (RT-DC) with Jochen Guck (Biotechnology Centre, TU Dresden).

The experiments described above have revealed the following main conclusions:

1) Loss of Cdc42 induces cell cannibalism through entosis, in a manner that depends upon cell transit through mitosis, and is associated with increased cortical RhoA activity.

2) Loss of Cdc42 is associated with decreased cell spreading and increased cell rounding during mitosis, which can be rescued by inhibition of the RhoA, ROCK and myosin axis. Cdc42 does not appear to operate through b1-integrin in this context.

3) Inhibition of Rap1 similarly enhances mitotic deadhesion and rounding, and induces subsequent entosis, indicating that these changes in mitotic morphology are sufficient to drive cell cannibalism.

4) The host cell can be wild type and fully adherent during mitotic entosis, raising the intriguing possibility that normal epithelial cells may engulf and destroy aberrantly proliferating neighbours, a potential means of tumour suppression.

Aims 3 and 4: Mitotic Entosis in Human Cancer and Chemotherapy

To investigate the occurrence and significance of mitotic entosis in human cancer, the strategies were undertaken:

1) In vitro and in vivo assays: Mitotic entosis was monitored using confocal and/or timelapse microscopy among the following: a) Human cancer cell lines, grown under adherent conditions, b) Human breast invasive ductal carcinoma cores (tissue microarray format, 75 samples), c) Mouse xenograft models (MCF7 human breast cancer cells). Cells were treated int eh presence or absence of Paclitaxel/taxol, a commonly used chemotherapeutic.

2) Cell fate: To assess cell fate following mitotic entosis, inner and outer cells were followed using timelapse and confocal microscopy. Inner cells were scored for death, and outer cells for multinucleation.

3) Oncogenes: In ongoing work, a variety of oncogenes (e.g. K-Ras V12, PI3K) have been analysed for their effects on cell cannibalism through entosis.

These experiments have yielded the following main conclusions:

1) Importantly, we find that mitotic entosis occurs basally among certain cancer cells in culture. Moreover, mitotic index positively correlates with frequency of cell cannibalism in primary tumour samples. These data indicate that mitosis, which is rife in a tumour, can drive cell cannibalism in cancer.

2) Mitotic entosis drives inner cells killing, a potentially tumour suppressive effect. On the other hand, this process also promotes outer cell multinucleation, which would be expected to drive aneuploidy and genomic instability in the longer term. As such, mitotic entosis has pleiotropic effects on cancer cell biology worthy of further investigation.

3) Paclitaxel/taxol treatment induces mitotic entosis in cultured cells and mouse xenograft models. These data reveal an unappreciated new function for this commonly used chemotherapeutic agents. Notably, Paclitaxel/taxol also enhances mitotic deadhesion and rounding, providing independent support for the link between changes in mitotic morphology and cell cannibalism through entosis.

Together, these studies have provided important new insights into the basic biology of both mitosis and cell cannibalism, which will inform these wider fields and open up interesting new lines of investigation for future research. Furthermore, this work establishes a clear link between mitosis and entosis in cancer and during chemotherapy, which is also worthy of further study. In future work, we will explore the translational potential of entosis in diagnosis, prognosis or prediction of therapeutic response. As such, this study has opened up new work with the potential for novel clinical impact.