Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis

The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer

PP1 initiates the dephosphorylation of MASTL, triggering mitotic exit and bistability in human cells

Entry into mitosis is driven by the phosphorylation of thousands of substrates, under the master control of Cdk1. During entry into mitosis, Cdk1, in collaboration with MASTL kinase, represses the activity of the major mitotic protein phosphatases, PP1 and PP2A, thereby ensuring mitotic substrates remain phosphorylated. For cells to complete and exit mitosis, these phosphorylation events must be removed, and hence, phosphatase activity must be reactivated. This reactivation of phosphatase activity presumably requires the inhibition of MASTL; however, it is not currently understood what deactivates MASTL and how this is achieved. In this study, we identified that PP1 is associated with, and capable of partially dephosphorylating and deactivating, MASTL during mitotic exit. Using mathematical modelling, we were able to confirm that deactivation of MASTL is essential for mitotic exit. Furthermore, small decreases in Cdk1 activity during metaphase are sufficient to initiate the reactivation of PP1, which in turn partially deactivates MASTL to release inhibition of PP2A and, hence, create a feedback loop. This feedback loop drives complete deactivation of MASTL, ensuring a strong switch-like activation of phosphatase activity during mitotic exit.CINSW FRL FellowshipThe Patricia Helen Guest FellowshipThe Petre Foundatio

Identification of PUMA as an estrogen target gene that mediates the apoptotic response to tamoxifen in human breast cancer cells and predicts patient outcome and tamoxifen responsiveness in breast cancer

Recognition of the pivotal role of estrogen in the aetiology of breast cancer has led to the development of antiestrogens (AE), such as tamoxifen (TAM) as effective therapies for the treatment and prevention of this disease. However, despite their widespread clinical efficacy, response to AEs is often short-lived, and acquired or innate therapeutic resistance remains a major obstacle in the successful treatment of breast cancer. Thus, delineating the intracellular pathways that mediate the cellular response to estrogen could potentially lead to new, more effective approaches to the treatment of breast cancer, particularly endocrine-resistant disease. Here, we have identified the BCL-2 homology 3 (BH3)-only, pro-apoptotic regulator, PUMA (p53 upregulated modulator of apoptosis) as an estrogen target gene that is acutely downregulated in response to estrogen in breast cancer cell lines, independently of their p53 status. PUMA is transcriptionally upregulated following treatment with TAM, and knock down of PUMA expression in these cells attenuates the apoptotic response to TAM. Furthermore, low PUMA expression in breast carcinomas is significantly associated with breast cancer-specific death (P0.0014 and P0.0115, for mRNA and protein, respectively), and worse outcome in TAM-treated patients (mRNA, P1.49e-05). These findings suggest that the dysregulation of apoptotic signaling pathways such as those executed through PUMA, can significantly impact on both the progression and therapeutic responsiveness of breast cancer. Moreover, they provide a convincing rationale for exploring new therapeutic approaches involving endocrine and non-endocrine therapies that target apoptotic pathways as an effective strategy for tackling endocrine refractory disease

PP1 initiates the dephosphorylation of MASTL, triggering mitotic exit and bistability in human cells

Entry into mitosis is driven by the phosphorylation of thousands of substrates, under the master control of Cdk1. During entry into mitosis, Cdk1, in collaboration with MASTL kinase, represses the activity of the major mitotic protein phosphatases, PP1 and PP2A, thereby ensuring mitotic substrates remain phosphorylated. For cells to complete and exit mitosis, these phosphorylation events must be removed, and hence, phosphatase activity must be reactivated. This reactivation of phosphatase activity presumably requires the inhibition of MASTL; however, it is not currently understood what deactivates MASTL and how this is achieved. In this study, we identified that PP1 is associated with, and capable of partially dephosphorylating and deactivating, MASTL during mitotic exit. Using mathematical modelling, we were able to confirm that deactivation of MASTL is essential for mitotic exit. Furthermore, small decreases in Cdk1 activity during metaphase are sufficient to initiate the reactivation of PP1, which in turn partially deactivates MASTL to release inhibition of PP2A and, hence, create a feedback loop. This feedback loop drives complete deactivation of MASTL, ensuring a strong switch-like activation of phosphatase activity during mitotic exit

5-Aza/E2 inhibits TAM-Wd cell proliferation.

<p>A. TAM-Wd and MCF-7 cell concentration response to E2 challenge (1×10⁻¹² M to 1×10⁻⁷ M) ±5-Aza (1×10⁻⁶ M). Cell counts were taken on day 7 of culture. Data shown represent E2-treated cell counts as a percentage of non-E2 treated control cells. Cell counts are significantly lower in 5-Aza treated TAM-Wd cells, co-treated with E2 at a concentration of 1×10⁻¹⁰ M and greater (*p<0.017). B. TAM-Wd cell concentration response to E2 challenge (1×10⁻¹² M to 1×10⁻⁷ M) +5-Aza (1×10⁻⁶ M) ±TAM (1×10⁻⁷ M). Cell counts were taken on day 7. Data shown represent E2-treated cell counts as a percentage of non-E2 treated control cells. The co-addition of TAM to 5-Aza treated TAM-Wd cells significantly changes the effect of E2 from a concentration of 1×10⁻⁹ M and greater (*p<0.026). C. Anchorage-dependent proliferation assay of TAM-Wd cells treated with E2 (1×10⁻⁹ M) ± TAM (1×10⁻⁷ M) in the presence of 5-Aza (1×10⁻⁶ M) for 14 days. The data shown represent actual cell number/well recorded over 3 independent experiments. By day 14, there are significantly more cells in the 5-Aza and 5-Aza/E2/TAM treated cells compared to TAM-Wd +5-Aza/E2 treated cells (*p<0.001). D. Cell cycle analysis using flow cytometric analysis of propidium iodide-stained TAM-Wd cells. Data represent percentage of cell in each phase relative to the total population.</p

5-Aza/E2 Co-treatment restored pS2 and PgR sensitivity to E2 challenge in the TAM-Wd cells.

<p>A. RT-qPCR evaluation of pS2 expression in MCF-7 and TAM-Wd cells ±5-Aza (1×10⁻⁶ M) ±E2 (1×10⁻⁹ M) for 48 hrs. Data shown represent percentage increase in pS2 detected in cells ±5-Aza following E2 challenge. Expression of pS2 is significantly increased in TAM-Wd cells treated with 5-Aza/E2 compared to 5-Aza treated cells (*p<0.001). Figure also shows ICC parallel analysis of pS2 protein expression in TAM-Wd cells (10× magnification) (representative of 3 independent experiments). B. RT-qPCR evaluation of PgR expression in MCF-7 and TAM-Wd cells ±5-Aza (1×10⁻⁶ M) ±E2 (1×10⁻⁹ M) for 48 hrs. Data shown represent percentage increase in PgR detected in cells ±5-Aza following E2 challenge. Expression of PgR is significantly increased in TAM-Wd cells treated with 5-Aza/E2 compared to 5-Aza treated cells (*p<0.001). Figure also shows ICC parallel analysis of PgR protein expression in TAM-Wd cells (10× magnification) (representative of 3 independent experiments). C. Clonal bisulphite sequencing analysis for the pS2 promoter region in TAM-Wd cells ±5-Aza (1×10⁻⁶ M) for 48 hrs. Colours and symbols are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040466#pone-0040466-g003" target="_blank">Figure 3</a>. D. Clonal bisulphite sequencing analysis for the PgR promoter region in TAM-Wd cells ±5-Aza (1×10⁻⁶ M) for 48 hrs. Colours and symbols are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040466#pone-0040466-g003" target="_blank">Figure 3</a>.</p