Research Lin28B In Breast Cancer Stem Cells And Mechanisms Of Regulation

Objectives:The incidence of breast cancer ranks first in all women malignant tumors in Western Europe, North America and other developed countries and is gradually raised in China. It was well known that cancer stem cells (CSCs) are highly tumorigenic, relatively resistant to conventional chemotherapy and radiotherapy and involved in both tumor initiation and metastasis. Tumor-promoting inflammation has been recognized as one of the hallmarks of cancer. Substantial evidence indicates that activation of the inflammatory pathway drives the self-renewal of CSCs; however, the molecular basis of this process remains undefined.Lin28is a RNA-binding protein and that activation of Lin28occurs in many different human tumors with a frequency of-15%. It has been reported that the repression of let-7by Lin28B could impair the self-renewal ability of breast CSCs. However, the mechanisms of crosstalk between Lin28and other signaling pathways in maintaining CSCs’function need to be further investigated.This study applied for a large number of clinical samples and in vitro/in vivo experiments to analysis the roles of Lin28B in human breast CSCs. Moreover, we studied the mechanism of Lin28B involving the IKKβ-mediated sternness maintenance in CSCs. Furthermore, we discussed IKKβ as therapeutic target to inhibit CSCs and tumor metastasis in vivo which could provide new idea and theoretical basis for cancer clinical treatment.Methods:Firstly, we tested the expression of Lin28B in breast cancer tissues by immunohistochemical and immunofluorescence staining to clear the effect of Lin28B in breast cancer progression and the correlation with ALDH1expression. Then, we confirmed the effect of Lin28B on physiological function of CSCs in vitro or in vivo. In vitro, the proportion of ALDH+tumor cells was detected by flow cytometry; the self-renewal ability of tumor cells was analyzed by tumorsphere culture; the invasion ability of tumor cells was detected by transwell experiment in human breast cancer cells MDA-MB-231. In vivo, two stable Lin28B silenced MDA-MB-231cell lines were established and then were transplanted into NOD/SCID mice; vernier caliper was used to measure the volume of tumor in mice; small animal in vivo imaging technology and H&E staining were used to analysis the pulmonary metastasis.Secondly, we studied the effect of inflammatory molecule IKKβ on maintaining CSCs’ function. The proportion of ALDH+cells were detection by flow cytometry; the self-renewal ability of tumor cells was analyzed by tumorsphere culture; the expression of sternness genes of Lin28B, SOX2, OCT4and Nanog were detected by RT-PCR or western blot. In vivo, small animal in vivo imaging technology and H&E staining were used to analysis the pulmonary metastasis in NOD/SCID mice. In order to further study the mechanism of IKKβ regulating Lin28B expression, related signaling pathways were screened; siRNA, Chromatin Immunoprecipitation and luciferase reporter assay were used to study the mechanism of TCF4regulating Lin28B expression.Furthermore, we studied the mechanism of Lin28B regulating Wnt signaling pathway with Lin28B knock-down MDA-MB-231cell line. TOPflash/FOPflash reporter system was used to detect the activity of Wnt signaling pathway; western blot, siRNA, and confocal were used to analyze the interaction of Lin28B with Wnt signaling pathway and its downstream target genes; RNA Immunoprecipitation was used to study the interaction between Lin28B protein and TCF4mRNA; immunohistochemical staining was used to detect the expression of TCF4in breast cancer tissues.Finally, we used the NOD/SCID mice model to study the therapeutical effect of IMD-0354combined with paclitaxel. Vernier caliper was used to measure the tumor volume in mice; small animal in vivo imaging technology and H&E staining were used to analyze the pulmonary metastasis; flow cytometry was used to detect the ALDH+tumor cells in tumor tissues; immunohistochemical staining was used to detect the expression of p-IKKβ, TCF4, Lin28B and ALDH1in tumor tissues.Results:1. Based on102cases of human breast cancer tissue samples and12normal cases adjacent to carcinoma, we found that Lin28B expression strongly correlated with clinical stages in human breast cancer patients and the stemness marker ALDH1.2. Silence of Lin28B expression in MDA-MB-231cells could significantly reduce the proportion of ALDH+cells, the tumorsphere forming ability and the invasion ability in vitro; Silence of Lin28B could significantly inhibit the growth of tumor cells and reduce the pulmonary metastasis in vivo.3. The higher IKKβ kinase activity was found in MDA-MB-231CSCs. Both IKKβ knockdown and activity inhibition could reduce the expression of Lin28B. IKKβ inhibitor IMD-0354could reduce the proportion of ALDH+cells, tumorsphere forming ability and invasion ability in MDA-MB-231cells. In vivo, IMD-0354could significantly inhibit the growth of tumor cells and reduce the pulmonary metastasis.4. Inhibition of IKKβ activity could impair the Wnt signaling pathway activity and reduce TCF4expression. Furthermore, TCF4could directly bind to the intron1of Lin28B gene to enhance the expression of Lin28B.5. Silence of Lin28B suppressed the activity of Wnt signaling pathway and reduced the TCF4and its downstream genes expression. Mechanically, Lin28B could directly interact with TCF4mRNA to promote its translation. Breast cancer tissue microarray staining revealed that TCF4expression was significantly correlated with Lin28B expression.6. IMD-0354combined with taxol could inhibit tumor growth and lung metastasis in NOD/SCID mice. Mechanically, IMD-0354could reduce the proportion of CSCs, and suppress the IKKβ/TCF4/Lin28B/ALDH1pathway.Conclusion:In this study, we observed that Lin28B expression was increased in breast cancer tissues and positively correlated with clinical progress in human breast cancer patients. Lin28B plays an important role in maintaining the stemness properties of CSCs and cell metastasis in human breast cancer cell. We also revealed a novel mechanism of the inflammatory gene IKKβ regulating stemness and identifying the TCF4/Lin28B loop as the pivotal molecular bridge connecting inflammation and stemness through their interaction with CSCs. This establishes a novel therapeutic strategy, with clinical potential, aimed at blocking activation of inflammation which targets breast CSC and suppresses tumor metastasis.