In the current study, we demonstrate that integrin α3β1 promotes invasive and metastatic traits of triple-negative breast cancer (TNBC) cells through induction of the transcription factor, Brain-2 (Brn-2). We show that RNAi-mediated suppression of α3β1 in MDA-MB-231 cells caused reduced expression of Brn-2 mRNA and protein and reduced activity of the BRN2 gene promoter. In addition, RNAi-targeting of Brn-2 in MDA-MB-231 cells decreased invasion in vitro and lung colonization in vivo, and exogenous Brn-2 expression partially restored invasion to cells in which α3β1 was suppressed. α3β1 promoted phosphorylation of Akt in MDA-MB-231 cells, and treatment of these cells with a pharmacological Akt inhibitor (MK-2206) reduced both Brn-2 expression and cell invasion, indicating that α3β1-Akt signaling contributes to Brn-2 induction. Analysis of RNAseq data from patients with invasive breast carcinoma revealed that high BRN2 expression correlates with poor survival. Moreover, high BRN2 expression positively correlates with high ITGA3 expression in basal-like breast cancer, which is consistent with our experimental findings that α3β1 induces Brn-2 in TNBC cells. Together, our study demonstrates a pro-invasive/pro-metastatic role for Brn-2 in breast cancer cells and identifies a role for integrin α3β1 in regulating Brn-2 expression, thereby revealing a novel mechanism of integrin-dependent breast cancer cell invasion.
Integrin α3β1, a cell adhesion receptor for certain laminins, is known to promote breast tumor growth and invasion. Our previous gene microarray study showed that the RELN gene, which encodes the extracellular glycoprotein Reelin, was upregulated in α3β1-deficient (i.e., α3 knockdown) MDA-MB-231 cells. In breast cancer, reduced RELN expression is associated with increased invasion and poor prognosis. In this study we demonstrate that α3β1 represses RELN expression to enhance breast cancer cell invasion. RELN mRNA was significantly increased upon RNAi-mediated α3 knockdown in two triple-negative breast cancer cell lines, MDA-MB-231 and SUM159. Modulation of baseline Reelin levels altered invasive potential, where enhanced Reelin expression in MDA-MB-231 cells reduced invasion, while RNAi-mediated suppression of Reelin in SUM159 cells increased invasion. Moreover, treatment of α3β1-expressing MDA-MB-231 cells with culture medium that was conditioned by α3 knockdown MDA-MB-231 cells led to decreased invasion. RNAi-mediated suppression of Reelin in α3 knockdown MDA-MB-231 cells mitigated this effect of conditioned-medium, identifying secreted Reelin as an inhibitor of cell invasion. These results demonstrate a novel role for α3β1 in repressing Reelin in breast cancer cells to promote invasion, supporting this integrin as a potential therapeutic target.
The development of integrin-targeted cancer therapies is hindered by incomplete understanding of integrin function in tumor cells and the tumor microenvironment. Previous studies showed that mice with epidermisspecific deletion of the a3 integrin subunit fail to form skin tumors during two-step chemical tumorigenesis, indicating a protumorigenic role for integrin a3b1. Here, we generated mice with tamoxifen-inducible, epidermis-specific a3 knockout to determine the role of a3b1 in the maintenance of established tumor cells and/or the associated stroma. Genetic ablation of a3 in established skin tumors caused their rapid regression, indicating that a3b1 is essential to maintain tumor growth. Although reduced proliferation and increased apoptosis were observed in a3b1-deficient tumor cells, these changes followed a robust increase in stromal apoptosis. Furthermore, macrophages and fibulin-2 levels were reduced in stroma following a3 deletion from tumor cells. Mass spectrometric analysis of conditioned medium from immortalized keratinocytes showed that a3b1 regulates a substantial fraction of the keratinocyte secretome, including fibulin-2 and macrophage CSF1; RNA in situ hybridization showed that expression of these two genes was reduced in tumor keratinocytes in vivo. Our findings identify a3b1 as a regulator of the keratinocyte secretome and skin tumor microenvironment and as a potential therapeutic target.
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Gene expression profiling, especially deep sequencing, has contributed significantly to our understanding of hair follicle stem cell biology; however, roles played by non-coding RNAs, especially the long non-coding RNAs (lncRNAs) for the most part remain undefined. We performed Illumina paired end sequencing and received raw reads for stem-and non-stem cell samples. FASTQC quality control was performed and adaptors were trimmed using Trimmomatic. We aligned the reads to the reference genome using Tophat. For mRNAs, we used USCS mm10 mouse annotated genome and GENCODE for lncRNA. Differential gene expression was calculated using DESeq between stem cell and non-stem cell. From DeSeq analysis, we found 2776 mRNA and 829 lncRNA that were statistically significant (Bonferroni p-value < 0.05 and fold change >2). Among the top-ranked genes, we found known keratinocyte stem cell genes (KSC): CD34, Keratin-15 (Krt15) and S100 family members. LncRNAs identified included known (Pvt1) and novel lncRNAs. Interestingly, we found an antisense lncRNA near a cluster of differentially expressed histone genes. To query function, we used parameters such as genomic location, co-expression analysis by Pearson coefficient correlation, and GO terms associated with co-expressed protein coding genes. After identifying the lncRNA pairs using bioinformatics, further biological validation involves RNA scope, qRTPCR, knock down of lncRNA-mRNA pairs in vitro, and in vivo effects on cell proliferation and carcinogenesis. These studies will determine the role of lncRNAs in hair follicle stem cells. Thus, we sequenced lncRNAS in conjunction with nearby mRNAs and compiled a unique data set focused on enriched mouse CD34+/CD49f+ hair follicle stem cells. We conclude that this approach furthers understanding of lncRNAs roles in KSCs and cancer, and will enable tissue specific patterns observed in human homologs of lncRNA-mRNA pairs to validate them as potential biomarkers and targets for treatments in mouse and human.
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