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Clarke, R. B.

Publications and source records attributed to Clarke, R. B..

2 recordsLinked to original sources
bioRxiv2019

Targeting endometrial cancer stem cell activity with metformin is inhibited by patient-derived adipocyte-secreted factors

Translational relevanceEndometrial cancer stem cells are reported to drive metastasis and disease relapse and are resistant to commonly used chemotherapeutic drugs. Here, we characterise ALDHhigh and CD133+ve endometrial cancer stem cells with differing levels of cancer stem cell activity and stem cell and epithelial-mesenchymal transition (EMT) gene expression. Their proportions and activity are reduced following treatment with metformin at a lower concentration than that required to affect bulk tumour cell proliferation, establishing a selective and specific effect on endometrial cancer stem cells. We demonstrate that this effect is abrogated when endometrial cancer cells are pre-treated with adipocyte conditioned media, indicating adipokine-mediated resistance to metformin. These results support future studies of metformin as an adjuvant therapy to reduce the risk of disease recurrence but should also investigate the impact of obesity on treatment response. AbstractO_ST_ABSPurposeC_ST_ABSAdvanced endometrial cancer continues to have a poor prognosis due to limited treatment options, which may be further adversely impacted by obesity. Endometrial cancer stem cells have been reported to drive metastasis, chemotherapy resistance and disease relapse, but have yet to be fully characterised and no specific targeted therapies have been identified. Here, we describe the phenotype and genotype of aldehyde dehydrogenase high (ALDHhigh) and CD133+ve endometrial cancer stem cells and how adipocyte secreted mediators block the inhibitory effect of metformin on endometrial cancer stem cell activity. Experimental designIshikawa and Hec-1a cell lines were used to characterise ALDHhigh and CD133+ve endometrial cancer cells using flow cytometry, functional sphere assays and quantitative-Polymerase Chain Reaction. The comparative effect of metformin on endometrial cancer stem cell activity and bulk tumour cell proliferation was determined using an Aldefluor and cytotoxicity assay. The impact of adipocyte secreted mediators on metformin response was established using patient-derived conditioned media. ResultsALDHhigh cells demonstrated greater endometrial cancer stem cell activity than CD133+ve cells and had increased expression of stem cell and epithelial-mesenchymal transition genes. Treatment with 0.5-1mM metformin reduced the proportion and activity of both endometrial cancer stem cell populations (p[≤]0.05), without affecting cell viability. This effect was, however, inhibited by exposure to patient-derived adipocyte conditioned media. ConclusionsThese results indicate a selective and specific effect of metformin on endometrial cancer stem cell activity, which is blocked by adipocyte secreted mediators. Future studies of metformin as an adjuvant therapy in endometrial cancer should be adequately powered to investigate the influence of body mass on treatment response.

cancer biology↗
bioRxiv2019

The milk protein alpha-casein suppresses triple negative breast cancer stem cell activity via STAT and HIF-1alpha signalling pathways in breast cancer cells and fibroblasts

Triple negative breast cancer (TNBC) is the most lethal breast cancer subtype. Extended periods of lactation protect against breast cancer development, but the mechanisms underlying this protection are unknown. We examined the effects of the milk protein alpha-casein over expression in the triple negative MDA-MB-231 breast cancer cell line. The effects of recombinant alpha-casein added exogenously to MDA-MB-231 breast cancer cells, and immortalised human fibroblasts were also investigated. We used transcriptional reporters to understand the signalling pathways downstream of alpha-casein in breast cancer cells and these fibroblasts that were activated by breast cancer cells. To extend our findings to the clinical setting, we analysed public gene expression datasets to further understand the relevance of these signalling pathways in triple negative breast cancer cells and patient samples. Finally, we used small molecular inhibitors to target relevant pathways and highlight these as potential candidates for the treatment of TN breast cancer. High levels of alpha-casein gene expression were predictive of good prognosis across 263 TNBC patient tumour samples. Alpha-casein over expression or exogenous addition reduces cancer stem cell (CSC) activity. HIF-1alpha was identified to be a key downstream target of alpha-casein, in both breast cancer cells and activated fibroblasts, and STAT transcription factors to be upstream of HIF-1alpha. Interestingly, HIF-1alpha is regulated by STAT3 in breast cancer cells, but STAT1 is the regulator of HIF-1alpha in activated fibroblasts. In analysis of 573 TNBC patient samples, alpha-casein expression, inversely correlated to HIF-1alpha, STAT3 and STAT1. STAT1 and STAT3 inhibitors target HIF-1alpha signalling in activated fibroblasts and MDA-MB-231 breast cancer cells respectively, and also abrogate CSC activities. Our findings provide an explanation for the protective effects of lactation in TNBC. Clinical data correlates high alpha-casein expression with increased recurrence-free survival in TNBC patients. Mechanistically, alpha-casein reduces breast cancer stem cell activity in vitro, and STAT3 and STAT1 were identified as regulators of pro-tumorigenic HIF-1alpha signalling in breast cancer cells and fibroblasts respectively.

cancer biology↗