The Study Of AURKA Protein Kinase In The Formation Of Vascular Mimicry In Triple-Negative Breast Cancer Stem Cells

Objective1. To isolate breast cancer stem cells from triple-negative breast cancer cells MDA-MB-231in serum-free suspension culture.To identify the character of breast cancer stem cells and observe the formation of vascular mimicry by three-dimensional culture.2. The expression of AURKA protein kinase from stem and normal cells were detected.AURKA protein kinase inhibitor were used to detect the ability of blood vessels formation and the expression of c-myc、sox-2、E-cadherin、β-catenin.3. In vivo the role of AURKA protein kinase in the formation of vascular mimicry in triple-negative breast cancer stem cells was confirmed using a xenograft-murine model of human triple-negative breast cancer.Methods1. The serum-free suspension culture of breast spheres from triple-negative breast cancer cells MDA-MB-231cells were isolated, the expression of stem cell transcription factor status of c-myc and sox-2were detected by RT PCR.2、The formation of vascular stem cells were obeserved by three-dimensional culture; The expression of AURKA protein kinase from stem and normal cells were detected by western blot; Three-dimensional culture and AURKA protein kinase inhibitor were used to detect the ability of blood vessels.RT PCR was used to detect the expression of c-myc、sox-2、E-cadherin、β-catenin in breast cancer stem cells and when AURKA protein kinase inhibitor were added.3. Female non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice5weeks old were used,We first isolate breast cancer stem cells.Breast cancer stem cells were collected,and cells were resuspended in1×106cells per100μl.cells were inoculated subcutaneously in the right flank.The mice were examined visually everyday. When tumor growth was measurable(10days after the injection),mice were weighed and tumors were measured with a caliper.Then mice were assigned into2groups(10mice each) receiving subcutaneous injection(100L of10%2-hydroxypropyl-cyclodextrin [Sigma-Aldrich]/1%sodium bicarbonate) or MLN8237(10mg/kg in a final formulation in10%2-hydroxypropyl--cyclodextrin/1%sodium bicarbonate) for10consecutive days.Throughout the study,the xenograft tumors were measured with a caliper everyday.Mice were euthanized after the10days treatment.The xenograft tumors were collected from mice immediately and fixed in4%formalin.To draw tumor growth curve based on the measured tumor volume,immunohistochemical and histochemical double-staining methods was used to detect the expression of c-myc、sox-2、E-cadherin、β-catenin and VM.Observe the effects of AURKA protein kinase inhibitor MLN-8237on the tumor.Result1. Cells initiated dividing into mammospheres compacted with several to decades cells after24h cultured.Incipient mammospheres were small,loose structure and morphous irregularity.Neogenesis dividing cells observed by inverted phase contrast microscope.Mammospheres halt augmentation after10to15d proliferation culture,appearance round or orbicular-ovate,bigger,cells connected tightly and margins between cells were ambiguious.The relative mRNA expression levels of c-myc、sox-2in breast cancer cells MDA-MB-231were significantly lower than breast cancer stem cells.2. Western blot was carried out to characterize AURKA protein kinase expression from triple-negative breast cancer cells MDA-MB-231and breast cancer stem cells. After action of serum-free suspension culture,the protein expression levels of AURKA from triple-negative breast cancer cells were significantly decreased compared with breast cancer stem cells.Breast cancer stem cells began to present spindle shape or polygon,to stretch out slender parapodium in24h when plated on Matrigel and form very characterized microvascular channels by48h(Figure3a).However,when AURKA protein kinase inhibitor were added to the t hree-dimensional culture,breast cancer stem cells aggregate and present clone shape.The formation of lumens structure was obviously reduced.The relative mRNA expression levels of c-myc、sox-2、E-cadherin、β-catenin in breast cancer stem cells were significantly lower when AURKA protein kinase inhibitor were added.3. Xenograft murine model results suggested that the speed of the tumor growth in administration treatment group was slower than control group, necrosis in cancer tissue is larger;The expression of c-myc、sox-2、β-catenin was higher in control group than administration treatment group, E-cadherin was higher in administration treatment group than control group. The presence of VM was much greater in control group than in treatment group.Conclusion1. Breast cancer stem cells were isolated from triple-negative breast cancer cells MDA-MB-231in serum-free suspension culture, the expression of c-myc、sox-2in breast cancer cells were significantly lower than breast cancer stem cells.2. The expression of AURKA from triple-negative breast cancer cells were significantly decreased compared with breast cancer stem cells, breast cancer stem cells could form characterized microvascular channels by three-dimensional culture. However,when AURKA protein kinase inhibitor were added, the formation of lumens structure was obviously reduced.AURKA protein kinase inhibitor could decrease the expression of c-myc、sox-2、E-cadherin、β-catenin in breast cancer stem cells.3. AURKA protein kinase inhibitor MLN8237obviously decreased the speed of the tumor growth in xenograft murine model,inhibited the presence of VM in cancer tissue,suggesting that AURKA protein kinase may become the new treatment target in suppressing angiogenesis and metastasis of triple-negative breast cancer.