HER2/Neu is overexpressed in 20-30% of breast cancers and associated with aggressive phenotypes and poor prognosis. For deciphering the role of HER2/Neu in breast cancer, mouse mammary tumor virus (MMTV)-Her2/neu transgenic mice that develop mammary tumors resembling human HER2-subtype breast cancer have been established. Several recent studies have revealed that HER2/Neu is overexpressed in and regulates self renewal of breast tumor initiating cells (TICs). However, in the MMTV-Her2/neu transgenic mouse model, the identity of TICs remains elusive, despite previous studies showing supportive evidence for existence of TICs in Her2/neu-induced mammary tumors. Through systematic screening and characterization, we identified surface markers CD49f, CD61 and ESA were aberrantly overexpressed in Her2-overexpressing mammary tumor cells. Analysis of these markers as well as CD24 detected anomalous expansion of the luminal progenitor population in preneoplastic mammary glands of Her2/neu-transgenic mice, indicating that aberrant luminal progenitors originated Her2-induced mammary tumors. The combined markers, CD49f and CD61, further delineated the CD49fhighCD61high-sorted fraction as a TIC-enriched population, which displayed increased tumorsphere formation ability, enhanced tumorigenicity both in vitro and in vivo and drug resistance to pacitaxel and doxorubicin. Moreover, the TIC-enriched population manifested increased TGFβ signaling and exhibited gene expression signatures of stemness, TGFβ signaling and Epithelial-to-Mesenchymal Transition. Our findings that self-renewal and clonogenicity of TICs were suppressed by pharmacologically inhibiting the TGFβ signaling further indicate that the TGFβ pathway is vital for maintenance of the TIC population. Finally, we showed that the integrin β3 (CD61) signaling pathway was required for sustaining active TGFβ signaling and self-renewal of TICs. We for the first time developed a technique to highly enrich TICs from mammary tumors of Her2/neu-transgenic mice, unraveled their properties and identified the cooperative integrin β3-TGFβ signaling axis as a potential therapeutic target for HER2-induced TICs.

A tumorsphere is a solid, spherical formation developed from the proliferation of one cancer stem/progenitor cell. These tumorspheres (Figure 1a) are easily distinguishable from single or aggregated cells (Figure 1b) as the cells appear to become fused together and individual cells cannot be identified. Cells are grown in serum-free, non-adherent conditions in order to enrich the cancer stem/progenitor cell population as only cancer stem/progenitor cells can survive and proliferate in this environment. This assay can be used to estimate the percentage of cancer stem/progenitor cells present in a population of tumor cells. The size, which can vary from less than 50 micrometers to 250 micrometers, and number of tumorspheres formed can be used to characterize the cancer stem/progenitor cell population within a population of in vitro cultured cancer cells and within in vivo tumors (Lo et al., 2012; Liu et al., 2009). While several cell lines can be used for tumorsphere formation assay (e.g. primary mammary tumor cells from Her2/neu-transgenic mice, MCF7, BT474 and HCC1954), some cell lines may not form typical tumorsphere structures and may be difficult to count or classify definitively as tumorspheres.

In this work we created electrospun fibrous scaffolds with random and aligned fiber orientations in order to mimic the 3D structure of the natural extracellular matrix (ECM). The rigidity and topography of the ECM environment have been reported to alter cancer cell behavior. But the complexity of the in vivo system makes it difficult to isolate and study such extracellular topographical cues that trigger cancer cells’ response. Breast cancer cells were cultured on these fibrous scaffolds for 3–5 days. The cells showed elongated spindle-like morphology in the aligned fibers whereas kept mostly flat stellar shape in the random fibers. Gene expression profiling of these cells post seeding, showed up-regulation of transforming growth factor β-1 (TGFβ-1) along with other mesenchymal biomarkers, suggesting that these cells are undergoing epithelial-mesenchymal transitions in response to the polymer scaffold. The results of this study indicate that the topographical cue may play a significant role in tumor progression.

Dysregulation of long non-codng RNA (lncRNA) expression has been found to contribute to tumorigenesis. However, the roles of lncRNAs in BRCA1-related breast cancer remain largely unknown. In this study, we delineate the role of the novel BRCA1/lncRNA NEAT1 signaling axis in breast tumorigenesis. BRCA1 inhibits NEAT1 expression potentially through binding to its genomic binding site upstream of the NEAT1 gene. BRCA1 deficiency in human normal/cancerous breast cells and mouse mammary glands leads to NEAT1 overexpression. Our studies show that NEAT1 upregulation resulting from BRCA1 deficiency stimulates in vitro and in vivo breast tumorigenicity. We have further identified molecular mediators downstream of the BRCA1/NEAT1 axis. NEAT1 epigenetically silences miR-129-5p expression by promoting the DNA methylation of the CpG island in the miR-129 gene. Silencing of miR-129-5p expression by NEAT1 results in upregulation of WNT4 expression, a target of miR-129-5p, which leads to activation of oncogenic WNT signaling. Our functional studies indicate that this NEAT1/miR-129-5p/WNT4 axis contributes to the tumorigenic effects of BRCA1 deficiency. Finally our in silico expression correlation analysis suggests the existence of the BRCA1/NEAT1/miR-129-5p axis in breast cancer. Our findings, taken together, suggest that the dysregulation of the BRCA1/NEAT1/miR-129-5p/WNT4 signaling axis is involved in promoting breast tumorigenesis.

Interdisciplinary approaches for molecular and cellular life sciences ISSN 1757-9694 www.rsc.org/ibiologyVolume 5 MDA-MB-231, by ALDEFLUOR assay and mammosphere formation assay. Moreover, we observed the upregulation of epithelial to mesenchymal transition and increased invasive capability in cells cultured on PCL fibrous scaffolds. These data suggest that the increase of CSC proportion in a 3D culture system may account for the enhanced malignancy. Therefore, our PCL fibrous scaffolds can potentially be used for CSCs enrichment and anti-cancer drug screening. Insight, innovation, integrationThree dimensional (3D) culture systems have been utilized in studies of cancer metastasis and anti-tumor drug screening. Accumulating evidence indicates that cancer cells in 3D culture displayed higher malignancy and invasive capability compared to their counterparts in two-dimensional culture. In the present paper, we have fabricated a 3D polycaprolactone fibrous scaffold using an electrospinning process to evaluate how CSCs proportion in breast cancer cell lines responds to this 3D culture. Functional assay and gene profiling together indicate that culture on fibrous scaffolds increased CSCs and induced epithelial-mesenchymal-transition. This work provides a better understanding of how a 3D culture condition affects cancer cells behavior as a mixture of CSCs and non-CSCs. Thus, this knowledge potentially enlightens the design of novel biomaterials for the enrichment of CSCs, and eventually for drug screening targeting CSCs.