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Li, C.
Doktoratsstudium der Medizinischen Wissenschaft; Humanmedizin; [ Dissertation ] Graz Medical University; 2015. pp.103. [OPEN ACCESS]


Authors Med Uni Graz:
Bauernhofer Thomas
Groschner Klaus
Schreibmayer Wolfgang

Perception of and reaction to mechanical stress is regarded to play meaningful roles in the lactating healthy mammary gland but also during cancerogenesis and metastasation of breast cancer. We investigated whether non-cancerous and cancerous breast cells possess functional MSCs within their plasma membranes, identified the molecular nature of the MSCs detected and assessed possible vital roles. The non-cancerous MCF-10A and the cancerous MCF-7 human cell lines were screened for MSCs with the patch clamp method, using cell-attached membrane patches. MSCs were detected in 157 out of 291 cell-attached membrane patches derived from MCF-7 (various kinds of pipette filling solution were used), being by far the most abundant ion channel species in this cell line under the conditions used (i.e. constant membrane potential; predominant ions within the patch pipette were Na+, K+ and Cl-). In contrast, MSCs were completely absent in MCF-10A cells under the same conditions (N=30). MSCs in MCF-7 were activated by negative pressure at the outer side of the membrane in a saturable manner (EP50: 41.2 ± 0.5 mbar (N=13)). Single channel conductance exerted to be 25.6 ± 0.4 pS (N=8) for 153 mmole/L K+. Permeability for monovalent cations exerted to be: Li+ < Na+ < K+ ˜ Rb+ ˜ Cs+. Divalent cations also permeated substantially with Ca2+ permeating better when compared to Ba2+. When a recently discovered MSC protein, human Piezo1 (hP1), was heterologously overexpressed in HEK-293 cells, ion channels with single channel conductances for Li+, K+ and Ca2+ that were indistinguishable from those observed in MCF-7 cells, were detected. Hence we conclude that MSCs in MCF-7 cells are formed by the hP1 protein. Due to a lack of an available antibody for detection of hP1 by Western Blots, quantitative RT-PCR was performed in order to assess hP1 mRNA expression levels. Messenger RNA encoding hP1 was present in MCF-10A but at significantly lower amounts when compared to MCF-7. When MCF-10A cells are transfected with plasmids encoding hP1, MSCs with single channel conductance indistinguishable to MSC in MCF-7 cells are observed. Motility of MCF-7 cells with and without GsMTx4, a tarantula toxin and specific blocker of MSCs, was monitored for 72 hours using the cell observer. Both motility and velocity of MCF-7 cells were reduced by GsMTx4. We conclude that hP1 protein acts as a mechanosensor in the plasma membrane of breast cancer cells controlling cellular motility. Possible roles in tumorigenesis and metastasation of breast cancer cells are indicated by the finding that the hazard ratio for overall survival is substantially increased upon hP1 mRNA overexpression in breast tumors (HR=1.63 (95% confidence interval: 1.26 – 2.09); p<0.00013; N=1115; KM Plotter ("" using the 2014 database), as described by Gyorffy et al., Breast Cancer Res Treatment, 123(3), 725-31, 2010).

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