Progression To Invasive Breast Cancer Research Articles

O025 Triggering mRNA stability switch by a miRNA inhibitor: Normalization of aberrant chemokine response in invasive breast cancer

Introduction Aberrant mRNA stability for a number of genes coding for AU-rich (ARE) mRNAs, including chemokines and growth factors, occurs in many cancer types. The CXCR4-CXCL12 (SDF-1 α ) axis is among the most deregulated chemokine responses that promote the progression of invasive breast cancer. Normalization of the aberrant CXCR4-CXCL12 response by triggering CXCR4 ARE-mRNA destabilization mediated by the zinc finger mRNA binding protein, tristetraprolin (TTP, ZFP36 ), is a potential and intriguing approach. Methods Several breast cancer cell lines were used for comparison with the MDA-MB-231 cell line as an invasive model of breast cancer. We treated the cells with a cell-permeable miR-29a inhibitor which triggers TTP expression, and monitored mRNA stability changes for key RNA binding proteins using RT-QPCR and reporter assays. RNA-immunoprecipitation was also employed to study the mRNA–protein interactions. Subsequent alterations in protein levels and associated functional attributes, including invasion and migration assays were investigated. Further, clinical data from a public database were used to understand these relationships. Results We found that CXCR-4 over-expression in invasive breast cancer is causally linked to deficient TTP expression. TTP deficiency was partly due to the high miR-29a levels that target TTP 3’UTR- this is observed in the highly-invasive MDA-MB-231 cells when compared to other non-invasive or non-tumorigenic cell lines. We also show that CXCR-4 is an mRNA target for TTP but not its zinc finger mutant, C124R, as the wild type was able to bind to CXCR-4 mRNA. TTP deficiency in MDA-MB-231 cells led to increased stability of mRNA coding for the RNA stabilizing protein, HuR, and subsequently HuR protein levels were increased. We demonstrated that HuR is able to bind to and increase CXCR-4 mRNA stability. Thus, the aberrant TTP-HuR balance results in CXCR-4 mRNA stabilization in invasive breast cancer. To overcome this aberration, we employed a cell-permeable peptide-linked miR-29a inhibitor to promote TTP mRNA upregulation and HuR mRNA down-regulation. Indeed this was the case, which subsequently led to CXCR-4 mRNA destabilization and reduction of CXCR-4 levels. This paralleled a reduction in the invasiveness of the cells and migration towards CXCR4 ligand, CXCL12. Clinical data showed that TTP-HuR mRNA ratios which reflect ARE-mRNA stability changes are perturbed in invasive breast cancer samples when compared to breast tissues ( p p Conclusion We establish that the chemokine receptor CXCR-4 is a novel mRNA target for TTP and HuR, and that its over-expression and breast cancer invasiveness is normalized by the miR29a inhibitor. The study shows that aberrant ARE-mediated mRNA stability in invasive breast cancer can be corrected by switching TTP-HuR balance towards mRNA destabilization in the CXCR4/CXCRL12 axis, indicating a potential therapeutic approach.

Regulation of DCIS to invasive breast cancer progression by Singleminded-2s (SIM2s)

Singleminded-2s (SIM2s) is a member of the bHLH/PAS family of transcription factors and a key regulator of mammary epithelial cell differentiation. SIM2s is highly expressed in mammary epithelial cells and down regulated in human breast cancer. Loss of Sim2s causes aberrant mouse mammary ductal development with features suggestive of malignant transformation, whereas over-expression of SIM2s promotes precocious alveolar differentiation in nulliparous mouse mammary glands, suggesting that SIM2s is required for establishing and enhancing mammary gland differentiation. To test the hypothesis that SIM2s regulates tumor cell differentiation, we analyzed SIM2s expression in human primary breast ductal carcinoma in situ (DCIS) samples and found that SIM2s is lost with progression from DCIS to invasive ductal cancer (IDC). Utilizing a MCF10DCIS.COM progression model, we have shown that SIM2s expression is decreased in MCF10DCIS.COM cells compared to MCF10A cells and reestablishment of SIM2s in MCF10DCIS.COM cells significantly inhibits growth and invasion in vitro and in vivo. Analysis of SIM2s-MCF10DCIS.com tumors showed that SIM2s promoted a more differentiated tumor phenotype including the expression of a broad range of luminal markers (CSN2 (β-casein), CDH1 (E-cadherin), and KER18 (keratin-18)) and suppressed genes associated with stem cell maintenance and a basal phenotype (SMO (smoothened), p63, SLUG (snail-2), KER14 (keratin-14) and VIM (vimentin)). Furthermore, loss of SIM2s expression in MCF10DCIS.COM xenografts resulted in a more invasive phenotype and increased lung metastasis likely due to an increase in hedgehog signaling and matrix metalloproteinase expression. Together, these exciting new data support a role for SIM2s in promoting human breast tumor differentiation and maintaining epithelial integrity.

Human primary ductal carcinoma in situ (DCIS) subtype‐specific pathology is preserved in a mouse intraductal (MIND) xenograft model

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer. The current recognition that DCIS lesions exhibit inter- and intra-lesion diversity suggests that the process of evolution to invasive breast cancer is more complex than previously recognized. Here we demonstrate the reproducible growth of primary DCIS cells derived from patient's surgical and biopsy samples by the mouse intraductal (MIND) model. MIND involves injection of cells into the NOD-SCID IL2Rgamma$^{{\rm{null}}}$ (NSG) mouse mammary ducts. Twelve (eight unique and four repeats) DCIS and two atypical hyperplasia specimens, heterogeneous with respect to biomarker expression and histology, were injected into 48 mouse mammary glands and analysed for successful xenotransplantation. Overall, 14/34 and 11/14 MIND xenotransplanted glands contained human DCIS and atypical hyperplastic cells, respectively, after 8 weeks, which formed single and multi-layered epithelium inside the ducts, and were heterogeneous with respect to expression of human cytokeratins, oestrogen receptor α (ER), and HER2. ER protein expression was recapitulated in MIND xenografts at ratios similar to the corresponding patient biopsies. In both patient biopsies and corresponding MIND xenografts, HER2 protein expression and nuclear HER2 gene overexpression were restricted to the DCIS lesions and were not found in the surrounding stroma or normal ducts. The xenografted DCIS lesions recapitulate the pathology and heterogeneity of human disease, thus providing a powerful tool for the characterization of the distinct cellular and molecular basis of inter- and intra-tumoural heterogeneity and the processes of DCIS to early invasive breast cancer progression.

Increased N-myc downstream-regulated gene 1 expression is associated with breast atypia-to-carcinoma progression

N-myc downstream-regulated gene-1 (NDRG1) has been identified as a protein involved in the differentiation of epithelial cells. As a newly metastasis suppressor gene, whether it contributes to carcinogenesis of breast cancer is still unknown. This study aimed to clarify the possible role of NDRG1 for breast cancer carcinogenesis, and further to investigate its clinicopathological significance in invasive breast cancer. We examined the expression of NDRG1 in normal epithelium of breast (n = 35), usual ductal hyperplasia (n = 22), atypical ductal hyperplasia (n = 33), atypical lobular hyperplasia (n = 8), ductal carcinoma in situ (n = 16), lobular carcinoma in situ (n = 6), invasive ductal carcinoma (n = 50), and invasive lobular carcinoma (n = 45) by immunohistochemistry and analyzed the correlation between NDRG expression and clinicopathological features of invasive breast cancer. Western blot analysis was carried out to investigate the expression of NDRG1 in 20 invasive ductal breast cancer and the paired non-tumor portion of the same case. NDRG1 expression in invasive breast cancer (70/95, 73.7%) was higher than that in noninvasive breast lesions (29/85, 34.1%; p < 0.05) which was higher than that in normal breast epithelium (5/35, 14.3%; p < 0.05). Statistical analysis revealed a significant correlation between NDRG1 expression with tumor stage in invasive breast cancer, and its expression in invasive ductal carcinoma is significantly higher than invasive lobular carcinoma (p < 0.05). It was not associated with age, menopausal status, tumor size, and lymph node metastasis. NDRG1 protein levels were significantly higher in invasive ductal breast cancer compared to the paired non-tumor portion of the same case by Western blot analysis (p < 0.05). Increased NDRG-1 expression is associated with breast atypia-to-carcinoma progression. NDRG1 expression might participate in the carcinogenesis and progression of invasive breast cancer. These findings provide further evidence that NDRG1 may serve as an important biomarker for invasive breast cancer.

Immunohistochemical expression of PTEN and survivin in ductal carcinoma of breast cancer and correlation with apoptotic index

Backround Survivin is a recently identified inhibitor of apoptosis and PTEN plays an important role in arrangement of signals required for cell growing and apoptosis. In case of absence or mutations of PTEN, tumour cells can protect themselves against apoptosis by the way of lipid signal transduction. Methods In this study, the relationship of PTEN and survivin expression with apoptosis was investigated by the means of immunohistochemical method in 39 patients with invasive breast cancer (IBC) and ductal carcinoma in situ (DCIS) within the same breast tissue. Results A decrease or loss in PTEN expression was observed in an important portion in DCIS (46.7%) and IBC (71.8%). In cases with invasive ductal carcinomas, strong staining with survivin was observed in 33.3% of cases, while there was weak staining in 61.6% and no staining in 5.1% of cases. All of the DCIS tumour cells were positive with survivin but staining was weak. Apoptotic index was higher in IBC than in DCIS, but there was no significant difference. No relationship was found between apoptosis and PTEN or survivin expression in breast carcinoma. Conclusion Our results suggest that a decrease in PTEN expression and increase in apoptosis is important in progression of IBC.