analysis-of-side-population-in-solid-tumor-cell-lines

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Nankai University

Gene expression profiling (GEP) had divided the diffuse large B-cell lymphoma (DLBCL) into molecular subgroups: germinal center B-cell like (GCB), activated B-cell like (ABC), and unclassified (UC) subtype. However, this classification with prognostic significance was not applied into clinical practice since there were more than 1000 genes to detect and interpreting was difficult. To classify cancer samples validly, eight significant genes (MYBL1, LMO2, BCL6, MME, IRF4, NFKBIZ, PDE4B, and SLA) were selected in 414 patients treated with CHOP/R-CHOP chemotherapy from Gene Expression Omnibus (GEO) data sets. Cutoffs for each gene were obtained using receiver-operating characteristic curves (ROC) new model based on the support vector machine (SVM) estimated the probability of membership into one of two subgroups: GCB and Non-GCB (ABC and UC). Furtherly, multivariate analysis validated the model in another two cohorts including 855 cases in all. As a result, patients in the training and validated cohorts were stratified into two subgroups with 94.0%, 91.0%, and 94.4% concordance with GEP, respectively. Patients with Non-GCB subtype had significantly poorer outcomes than that with GCB subtype, which agreed with the prognostic power of GEP classification. Moreover, the similar prognosis received in the low (0-2) and high (3-5) IPI scores group demonstrated that the new model was independent of IPI as well as GEP method. In conclusion, our new model could stratify DLBCL patients with CHOP/R-CHOP regimen matching GEP subtypes effectively.

Machine learning-based classification of diffuse large B-cell lymphoma patients by eight gene expression profiles.

Long non-coding RNAs are known to be involved in cancer progression, but their biological functions and prognostic values are still largely unexplored in diffuse large B-cell lymphoma. In this study, long non-coding RNAs expression was characterized in 1,403 samples including normal and diffuse large B-cell lymphoma by repurposing 7 microarray datasets. Compared with any stage of normal B cells, NONHSAG026900 expression was significantly decreased in tumor samples. And in germinal center B-cell subtype, the significantly higher expression of NONHSAG026900 indicated it was a favorable prognosis biomarker. Then the prognostic power of NONHSAG026900 was validated with another independent dataset and NONHSAG026900 improved the predictive power of International Prognostic Index as an independent factor. Moreover, functional prediction and validation demonstrated that NONHSAG026900 could inhibit cell cycle activity to restrain tumor proliferation. These findings identified NONHSAG026900 as a novel prognostic biomarker and offered a new therapeutic target for diffuse large B-cell lymphoma patients.

The long non-coding RNA NONHSAG026900 predicts prognosis as a favorable biomarker in patients with diffuse large B-cell lymphoma.

Fos-related antigen 1 (Fra1) has been proposed as a gatekeeper of the mesenchymal-epithelial transition to epithelial-mesenchymal transition. Here, we showed that de-phosphorylated JNK2 increased the expression of Fra1 by promoting the expression of c-Jun and Jun-B. Conversely, phosphorylated JNK2 suppressed its expression via enhancing the ubiquitination of c-Jun and Jun-B. These data provided insights into the regulatory mechanism of JNK2 on the expression of Fra1. Our study thus demonstrated that the conversion of JNK2 from its phosphorylation to de-phosphorylation status promoted the switch of breast cancer cells from mesenchymal-epithelial transition to epithelial-mesenchymal transition.

Alternative promotion and suppression of metastasis by JNK2 governed by its phosphorylation.

As an important component of the tumor microenvironment, CD4 CD25 Tregs reduce antitumor immunity, promote angiogenesis and metastasis in breast cancer. However, their function in regulating the "stemness" of tumor cells and the communication between Tregs and cancer stem cells (CSCs) remain elusive. Here, we disclose that the primarily cultured Tregs isolated from breast-tumor-bearing Foxp3-EGFP mouse upregulate the stemness property of breast cancer cells. Tregs increased the side-population and the Aldehyde dehydrogenase-bright population of mouse breast cancer cells, promoted their sphere formation in a paracrine manner, and enhanced the expression of stemness genes, such as Sox2 and so forth. In addition, Tregs increased tumorigenesis, metastasis, and chemoresistance of breast cancer cells. Furthermore, Sox2-overexpression tumor cells activated NF-κB-CCL1 signaling to recruit Tregs through reducing the binding of H3K27Me3 on promoter regions of p65 and Ccl1. These findings reveal the functional interaction between Tregs and CSCs and indicate that targeting on the communication between them is a promising strategy in breast cancer therapy. Stem Cells 2017;35:2351-2365.

Sox2 Communicates with Tregs Through CCL1 to Promote the Stemness Property of Breast Cancer Cells.

Current predictive model is not developed by inflammation-related genes to evaluate clinical outcome of breast cancer patients.

Three inflammation-related genes could predict risk in prognosis and metastasis of patients with breast cancer.

 Bone is one of the most common metastatic sites of breast cancer. CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor superfamily, is mainly expressed in activated leukocytes. Previous study demonstrates the effect of CD137-CD137L bidirectional signaling pathway on RANKL-mediated osteoclastogenesis. However, the role of CD137 in bone metastasis of breast cancer needs further study.  Stable monocyte/macrophage cell lines with  overexpression and silencing were established. Western blot, real-time PCR, transwell and tartrate-resistant acid phosphatase staining were used to detect the regulatory effect of CD137 on migration and osteoclastogenesis of monocytes/macrophages . Spontaneous bone metastasis mouse model was established, bioluminescent images, immunohistochemistry and histology assay were performed to detect the function of CD137 in bone metastasis .  We found that CD137 promotes the migration of monocytes/macrophages to tumor microenvironment by upregulating the expression of Fra1. It also promoted the differentiation of monocytes/macrophages into osteoclasts at the same time, thus providing a favorable microenvironment for the colonization and growth of breast cancer cells in bone. Based on these findings, a novel F4/80-targeted liposomal nanoparticle encapsulating the anti-CD137 blocking antibody (NP-αCD137 Ab-F4/80) was synthesized. This nanoparticle could inhibit both bone and lung metastases of 4T1 breast cancer cells with high efficacy . In addition, it increased the therapeutic efficacy of Fra1 inhibitor on tumor metastasis.  Taken together, these findings reveal the promotion effect of macrophage/monocyte CD137 on bone metastases and provide a promising therapeutic strategy for metastasis of breast cancer.

CD137 promotes bone metastasis of breast cancer by enhancing the migration and osteoclast differentiation of monocytes/macrophages.

Metastases are the main cause of cancer-related mortality in breast cancer. Although significant progress has been made in the field of tumor metastasis, the exact molecular mechanisms involved in tumor metastasis are still unclear. Here, we report that ATOH8-V1, a novel isoform of ATOH8, is highly expressed in breast cancer and is a negative prognostic indicator of survival for patients. Forced expression of ATOH8-V1 dramatically enhances, while silencing of ATOH8-V1 decreases the metastasis of breast cancer cell lines. Moreover, ATOH8-V1 directly binds to the RhoC promoter and stimulates the expression of RhoC, which in turn enhances the metastasis of breast cancer. Altogether, our data demonstrate that ATOH8-V1 is a novel pro-metastatic factor that enhances cancer metastasis, suggesting that ATOH8-V1 is a potential therapeutic target for treatment of metastatic cancers.

A novel isoform of ATOH8 promotes the metastasis of breast cancer by regulating RhoC.

 Tumor microenvironment interacts with tumor cells to regulate their stemness properties through various cytokines and cytokine receptors. Previous studies revealed the possible role of interleukin 20 receptor subunit alpha (IL20RA) signaling in the progression of several types of tumors. However, its regulatory effects on the stemness and the microenvironment of breast cancer need to be studied.  Immunohistochemical staining and western blot analysis were used to evaluate the association between IL20RA and SOX2 in breast tumors and noncancerous tissues. Enzyme-linked immunosorbent assay and TCGA dataset analysis were performed to determine the function of IL20RA signaling in breast cancer progression. Gain- and loss-of-function methods were performed to examine the effects of IL20RA on the stemness of breast cancer cells. The stemness features were analyzed by detecting the expression of core stemness genes, side population (SP), sphere formation ability, and aldehyde dehydrogenase (ALDH) activity. Flow cytometric analysis was applied to detect the changes of tumor-infiltration lymphocytes in tumor tissues in mice. Based on the relevant molecular mechanisms elucidated in this study, a novel IL20RA-targeted liposomal nanoparticle encapsulating the signal transducer and activator of transcription 3 (STAT3) inhibitor stattic (NP-Stattic-IL20RA) was synthesized. These NPs were combined with anti-programmed death ligand 1 (PD-L1) antibody and chemotherapy to inhibit the development of breast tumors in mice.  IL20RA is highly expressed in human breast cancers and is positively associated with the SOX2 expression. IL20RA increases the SP and ALDH proportions of breast cancer cells, enhances the sphere formation ability, and promotes the expression of core stemness genes, such as  and , as well as increases chemoresistance of breast cancer cells. IL20RA promotes the tumor-initiating ability and lung metastasis of breast cancer cells . In addition, IL20RA activates the Janus kinase 1 (JAK1)-STAT3-SOX2 signaling pathway, leading to increased expression of PD-L1 and reduced recruitment of anti-cancer lymphocytes, including CD8 T cells and natural killer cells. Meanwhile, IL20RA signaling enhances the proportion of myeloid-derived suppressor cells. Combined with anti-PD-L1 antibody and NPs-Stattic-IL20RA, the chemotherapeutic efficacy was increased in breast cancer mouse models .  Collectively, our results reveal that the IL20RA pathway is a novel signaling pathway involved in promoting the stemness features of breast cancer along with the formation of a tumor-favorable immune microenvironment. Targeting the IL20RA population with STAT3 signaling inhibition combined with anti-PD-L1 antibody can increase the therapeutic efficacy of chemotherapeutic agents for breast cancer. This study thus introduces a promising novel strategy for breast cancer therapy.

Article	Year
Machine learning-based classification of diffuse large B-cell lymphoma patients by eight gene expression profiles. Cancer medicine\| PubMed ID: 26869285	2016
The long non-coding RNA NONHSAG026900 predicts prognosis as a favorable biomarker in patients with diffuse large B-cell lymphoma. Oncotarget\| PubMed ID: 28423735	2017
Alternative promotion and suppression of metastasis by JNK2 governed by its phosphorylation. Oncotarget\| PubMed ID: 28915613	2017
Sox2 Communicates with Tregs Through CCL1 to Promote the Stemness Property of Breast Cancer Cells. Stem cells (Dayton, Ohio)\| PubMed ID: 29044882	2017
Three inflammation-related genes could predict risk in prognosis and metastasis of patients with breast cancer. Cancer medicine\| PubMed ID: 30632703	2019
CD137 promotes bone metastasis of breast cancer by enhancing the migration and osteoclast differentiation of monocytes/macrophages. Theranostics\| PubMed ID: 31244935	2019
A novel isoform of ATOH8 promotes the metastasis of breast cancer by regulating RhoC. Journal of molecular cell biology\| PubMed ID: 33049034	2020
IL20RA signaling enhances stemness and promotes the formation of an immunosuppressive microenvironment in breast cancer. Theranostics\| PubMed ID: 33456560	2021

Xiaoli Dong

Biography

JoVE Journal Publications

Other Publications