Cd147 Reprograms Breast Cancer Cells Under Anoikis-inducing Conditions

Background: As an important characteristic of malignant tumors, metastasis causes around 90% of death in cancer patients. At present, it is considered that there is a small group of cells in tumor tissue, which called cancer stem cells(CSCs), has the ability of initiating tumor formation and growth. And, it is CSCs that cause tumor metastasis and recurrence in clinical patients. Nowadays, numerous evidences demonstrated that CSCs exist in many kinds of blood and solid tumors. Breast cancer ranks first in newly discovered cancer in women, and the second commonest cause of cancer-related death, which poses a serious threat to the health and life quality of women. It is metastasis that responsible for the vast majority of death in breast cancer patients. Breast CSCs is the first one to be reported among solid tumor stem cells, and may be the source of breast cancer metastasis. Therefore, a study on the cellular and molecular mechanisms of breast CSCs contributing to the metastasis is of great importance to a targeted repression of recurrence and metastasis in breast cancer. Numerous studies have suggested that CSCs, which can induce tumor metastasis, possessed of some intrinsic characteristics, such as anoikis resistance. In order to complete metastasize, tumor cells need to overcome the stress pressure that brought by anoikis. When facing with a variety of stresses, tumor cells themselves initiative active response to combat them. For example, current researches on CSCs shows that non-CSCs could be reprogrammed into CSCs reacting to stresses such as chemotherapy and radiotherapy, and then survived and metastasized. Anoikis is also a kind of stress pressure. Previou studies are mainly focused on enriching CSCs upon detachment, while pay little attention on the effects of detachment on the CSCs. Our study aims to explore the impact of detachment on CSCs and the corresponding cellular and molecular mechanism.Methods: This study was divided into two parts. In the first part, the anoikis was induced by culturing breast cancer cells under ultra-low adsorption plates. The ratio and function of breast CSCs cultivated under suspension or adherent conditions was analyzed by flow cytometry and tumor microsphere culture. Cancer cells with or without stem cell property was sorted by flow cytometry and cultured under suspension or adherent conditions, then the ratio of CSCs was further compared. In the second part, we have detected the expression of CD147 in anoikis resistant cells, and then constructed the cells with CD147 stable knock-down. We analyzed the in vitro proliferation and sphere-forming ability in CD147 knock-down and control cells by colony formation and tumorsphere formation. The ratio of breast CSCs in CD147 knock-down and control cells cultured under suspension or adherent conditions was also compared by flow cytometry. And, we also monitored the ratio of breast CSCs in CD147 over-expressed cells cultured under suspension or adherent conditions. Finally, we used western blot to detect the activation of STAT3 signaling pathway under detachment.Results: We found that the percentage of breast CSCs and its ability of forming microsphere under detachment, either from breast cancer cell lines or from malignant pleural effusion, were all significantly elevated as compared to adherent culture conditions. Non-CSCs sorted by flow cytometry reprogrammed into CSCs while growing under suspension system. Besides, membrane CD147 expression in the breast cancer cells was significantly increased upon detachment. Both in vitro proliferation and tumorsphere forming abilities were reduced in CD147 knock-down as compared with control cells. Furthermore, CD147 knock-down greatly abolished the reprogramming of non-CSCs to CSCs; while CD147 knock-in facilitated the increase of the ratio of CSCs induced by detachment. Preliminary research on molecular mechanism suggested that STAT3-Bcl-x L signaling pathway, the CD147 downstream signaling, was activated upon detachment.Conclusion: Detachment can lead to non-CSCs reprograming into CSCs, during which process the expression of CD147 was up-regulated. CD147 knock-down inhibited the increase of CSCs numbers induced by detachment; while CD147 knock-in significantly facilitate the above enhancement, which indicating that CD147 involved in the detachment-induced breast cancer cells reprogramming. And, the activation of CD147 downstream signaling, that is STAT3-Bcl-x L pathway, may play a role in this reprogramming process.