Gang Tu

Cancer-associated fibroblasts: A multifaceted driver of breast cancer progression

Cancerous tissue is a complex mix of tumor cells, stromal cells and extracellular matrix (ECM), all of which make up a disordered and aggressive niche in comparison with organized and homeostatic normal tissue. It is well accepted that the tumor microenvironment plays an indispensable role in cancer development, and thus can be recognized as an additional cancer hallmark alongside those that are well established. In breast cancer, cancer associated fibroblasts (CAFs) are the predominant cellular components and play a centric role in the tumor microenvironment since they not only promote cancer initiation, growth, invasion, metastasis and therapeutic resistance but are also involved in microenvironmental events including angiogenesis/lymphangiogenesis, ECM remodeling, cancer-associated inflammation and metabolism reprogramming, all of which are known to have pre-malignancy potency. At the molecular level, there is a sophisticated network underlying the interactions between CAFs and epithelial cells as well as other stromal components. Accordingly, targeting CAFs provides a novel strategy in cancer therapy. Herein, we summarize the current understanding of the role of CAFs in breast cancer.

GPR30 as an initiator of tamoxifen resistance in hormone-dependent breast cancer

IntroductionTamoxifen is widely used to treat hormone-dependent breast cancer, but its therapeutic benefit is limited by the development of drug resistance. Here, we investigated the role of estrogen G-protein coupled receptor 30 (GPR30) on Tamoxifen resistance in breast cancer.MethodsPrimary tumors (PTs) of breast cancer and corresponding metastases (MTs) were used to evaluate the expression of GPR30 and epidermal growth factor receptor (EGFR) immunohistochemically. Tamoxifen-resistant (TAM-R) subclones derived from parent MCF-7 cells were used to investigate the role of GPR30 in the development of tamoxifen resistance, using MTT assay, western blot, RT-PCR, immunofluorescence, ELISA and flow cytometry. TAM-R xenografts were established to assess anti-tumor effects of combination therapy with GPR30 antagonist G15 plus 4-hydroxytamoxifen (Tam), using tumor volume measurement and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).ResultsIn 53 human breast cancer specimens, GPR30 expression in MTs increased compared to matched PTs; in MTs, the expression patterns of GPR30 and EGFR were closely related. Compared to parent MCF-7 cells, TAM-R cells had greater growth responses to 17β-estradiol (E2), GPR30 agonist G1 and Tam, and significantly higher activation of Mitogen-activated protein (MAP) kinases; but this increased activity was abolished by G15 or AG1478. In TAM-R cells, GPR30 cell-surface translocation facilitated crosstalk with EGFR, and reduced cAMP generation, attenuating inhibition of EGFR signaling. Combination therapy both promoted apoptosis in TAM-R cells and decreased drug-resistant tumor progression.ConclusionsLong-term endocrine treatment facilitates the translocation of GPR30 to cell surfaces, which interferes with the EGFR signaling pathway; GPR30 also attenuates the inhibition of MAP kinases. These factors contribute to tamoxifen resistance development in breast cancer. Combination therapy with GPR30 inhibitors and tamoxifen may provide a new therapeutic option for drug-resistant breast cancer.

LncRNA-Hh Strengthen Cancer Stem Cells Generation in Twist-Positive Breast Cancer via Activation of Hedgehog Signaling Pathway.

Cancer stem cells (CSCs) are a subpopulation of neoplastic cells with self‐renewal capacity and limitless proliferative potential as well as high invasion and migration capacity. These cells are commonly associated with epithelial‐mesenchymal transition (EMT), which is also critical for tumor metastasis. Recent studies illustrate a direct link between EMT and stemness of cancer cells. Long non‐coding RNAs (lncRNAs) have emerged as important new players in the regulation of multiple cellular processes in various diseases. To date, the role of lncRNAs in EMT‐associated CSC stemness acquisition and maintenance remains unclear. In this study, we discovered that a set of lncRNAs were dysregulated in Twist‐positive mammosphere cells using lncRNA microarray analysis. Multiple lncRNAs‐associated canonical signaling pathways were identified via bioinformatics analysis. Especially, the Shh‐GLI1 pathway associated lncRNA‐Hh, transcriptionally regulated by Twist, directly targets GAS1 to stimulate the activation of hedgehog signaling (Hh). The activated Hh increases GLI1 expression, and enhances the expression of SOX2 and OCT4 to play a regulatory role in CSC maintenance. Thus, the mammosphere‐formation efficiency (MFE) and the self‐renewal capacity in vitro, and oncogenicity in vivo in Twist‐positive breast cancer cells are elevated. lncRNA‐Hh silence in Twist‐positive breast cells attenuates the activated Shh‐GLI1 signaling and decreases the CSC‐associated SOX and OCT4 levels, thus reduces the MFE and tumorigenesis of transplanted tumor. Our results reveal that lncRNAs function as an important regulator endowing Twist‐induced EMT cells to gain the CSC‐like stemness properties. Stem Cells 2016;34:55–66

Acquisition of epithelial-mesenchymal transition phenotype in the tamoxifen-resistant breast cancer cell: a new role for G protein-coupled estrogen receptor in mediating tamoxifen resistance through cancer-associated fibroblast-derived fibronectin and β1-integrin signaling pathway in tumor cells.

IntroductionAcquired tamoxifen resistance remains the major obstacle to breast cancer endocrine therapy. β1-integrin was identified as one of the target genes of G protein-coupled estrogen receptor (GPER), a novel estrogen receptor recognized as an initiator of tamoxifen resistance. Here, we investigated the role of β1-integrin in GPER-mediated tamoxifen resistance in breast cancer.MethodsThe expression of β1-integrin and biomarkers of epithelial-mesenchymal transition were evaluated immunohistochemically in 53 specimens of metastases and paired primary tumors. The function of β1-integrin was investigated in tamoxifen-resistant (MCF-7R) subclones, derived from parental MCF-7 cells, and MCF-7R β1-integrin-silenced subclones in MTT and Transwell assays. Involved signaling pathways were identified using specific inhibitors and Western blotting analysis.ResultsGPER, β1-integrin and mesenchymal biomarkers (vimentin and fibronectin) expression in metastases increased compared to the corresponding primary tumors; a close expression pattern of β1-integrin and GPER were in metastases. Increased β1-integrin expression was also confirmed in MCF-7R cells compared with MCF-7 cells. This upregulation of β1-integrin was induced by agonists of GPER and blocked by both antagonist and knockdown of it in MCF-7R cells. Moreover, the epidermal growth factor receptor/extracellular regulated protein kinase (EGFR/ERK) signaling pathway was involved in this transcriptional regulation since specific inhibitors of these kinases also reduced the GPER-induced upregulation of β1-integrin. Interestingly, silencing of β1-integrin partially rescued the sensitivity of MCF-7R cells to tamoxifen and the α5β1-integrin subunit is probably responsible for this phenomenon. Importantly, the cell migration and epithelial-mesenchymal transition induced by cancer-associated fibroblasts, or the product of cancer-associated fibroblasts, fibronectin, were reduced by knockdown of β1-integrin in MCF-7R cells. In addition, the downstream kinases of β1-integrin including focal adhesion kinase, Src and AKT were activated in MCF-7R cells and may be involved in the interaction between cancer cells and cancer-associated fibroblasts.ConclusionsGPER/EGFR/ERK signaling upregulates β1-integrin expression and activates downstream kinases, which contributes to cancer-associated fibroblast-induced cell migration and epithelial-mesenchymal transition, in MCF-7R cells. GPER probably contributes to tamoxifen resistance via interaction with the tumor microenvironment in a β1-integrin-dependent pattern. Thus, β1-integrin may be a potential target to improve anti-hormone therapy responses in breast cancer patients.

GPER mediates enhanced cell viability and motility via non-genomic signaling induced by 17β-estradiol in triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is an aggressive breast cancer with a generally poor prognosis. Due to lack of specific targets for its treatment, an efficient therapy is needed. G protein-coupled estrogen receptor (GPER), a novel estrogen receptor, has been reported to be expressed in TNBC tissues. In this study, we investigated the effects of blocking non-genomic signaling mediated by the estrogen/GPER pathway on cell viability and motility in the TNBC cells. GPER was strongly expressed in the TNBC cell lines MDA-MB-468 and MDA-MB-436, and the estrogen-mediated non-genomic ERK signaling activated by GPER was involved in cell viability and motility of TNBC cells. Treatment with 17β-estradiol (E2), the GPER-specific agonist G-1 and tamoxifen (TAM) led to rapid activation of p-ERK1/2, but not p-Akt. Moreover, estrogen/GPER/ERK signaling was involved in increasing cell growth, survival, and migration/invasion by upregulating expression of cyclinA, cyclinD1, Bcl-2, and c-fos associated with the cell cycle, proliferation, and apoptosis. Immunohistochemical analysis of TNBC specimens showed a significantly different staining of p-ERK1/2 between GPER-positive tissues (58/66, 87.9%) and GPER-negative tissues (13/30, 43.3%). The positivity of GPER and p-ERK1/2 displayed a strong association with large tumor size and poor clinical stage, indicating that GPER/ERK signaling might also contribute to tumor progression in TNBC patients which corresponded with in vitro experimental data. Our findings suggest that inhibition of estrogen/GPER/ERK signaling represents a novel targeted therapy in TNBC.

LncRNA expression signatures of twist-induced epithelial-to-mesenchymal transition in MCF10A cells.

The epithelial-to-mesenchymal transient (EMT) is associated with tumor metastasis. Twist is one of the key transcription factors for EMT and relates to tumor cell migration. Long non-coding RNAs (lncRNAs) have recently emerged as important regulatory molecules involved in a broad range of biological processes and complicated diseases. However, it is unknown whether a signal network and lncRNAs are involved in Twist-induced EMT program. Taking MCF10A/Twist as a model, more than 99 lncRNAs and 3164 genes are regulated in the Twist-induced EMT process using lncRNA-array and cDNA micro-array. We establish a downstream signal network associated with EMT induced by Twist using bioinformatic analysis (Gene Ontology, pathway analysis) and experimental data. A set of multiple canonical signal pathways (such as WNT, MAPK, JAK/STAT, TGF-β, mTOR, Hedgehog and P53 signaling pathways) and several lncRNAs [such as lncRNA (chr6, 26124411-26139312, +), lncRNA (chr1, 41944445-41949874, -), lncRNA (chr17, 44833874-44834830, +)] are altered in MCF10A/Twist cells. More interestingly, lncRNA (chr17, 44833874-44834830, +), lncRNA (chr17, 21142183-21156578, -), lncRNA (chr6, 26124411-26139312, +) and lncRNA (chr19, 438420-2083745, -) may be involved in regulation or activation of WNT signaling pathway in the Twist-induced EMT process. These findings first determine that Twist contributes to invasion and metastasis by inducing wide-ranging transcriptional and functional changes of lncRNAs and signal pathways in our study.

G-protein Coupled Estrogen Receptor 1 Expression in Primary Breast Cancers and Its Correlation with Clinicopathological Variables

Purpose G-protein coupled estrogen receptor 1 (GPER) probably play important roles in the progression of breast cancer including endocrine therapeutic resistance. We evaluated GPER in primary breast cancers. Methods Immunohistochemistry was used to detect GPER in paraffin-embedded tissues of primary breast cancers from 423 patients and GPER expression was correlated with clinicopathological factors. Results GPER was expressed in 63.8% of specimens, coexpressed with estrogen receptor alpha (ERα) in 36.6% of tumors and was positive in 62.5% of the ERα-negative tumors. The expression of GPER had no relationship with the status of ERα, progesterone receptor and HER2. Although the expression of GPER was significantly inversely related with nodal status (p=0.045), no correlation between GPER expression and other clinicopathological variables (age, menstruation status, tumor size, stage, histologic grade, Nottingham Prognostic Index or pathological type) was found. Conclusion GPER and ERα exhibited independent expression pattern of distribution in primary breast cancers. A long-term follow-up and a more definite molecular phenotype for ER are necessary in confirming studies.

Oxidized ATM promotes abnormal proliferation of breast CAFs through maintaining intracellular redox homeostasis and activating the PI3K-AKT, MEK-ERK, and Wnt-β-catenin signaling pathways

Abnormal proliferation is one characteristic of cancer-associated fibroblasts (CAFs), which play a key role in tumorigenesis and tumor progression. Oxidative stress (OS) is the root cause of CAFs abnormal proliferation. ATM (ataxia-telangiectasia mutated protein kinase), an important redox sensor, is involved in DNA damage response and cellular homeostasis. Whether and how oxidized ATM regulating CAFs proliferation remains unclear. In this study, we show that there is a high level of oxidized ATM in breast CAFs in the absence of double-strand breaks (DSBs) and that oxidized ATM plays a critical role in CAFs proliferation. The effect of oxidized ATM on CAFs proliferation is mediated by its regulation of cellular redox balance and the activity of the ERK, PI3K-AKT, and Wnt signaling pathways. Treating cells with antioxidant N-acetyl-cysteine (NAC) partially rescues the proliferation defect of the breast CAFs caused by ATM deficiency. Administrating cells with individual or a combination of specific inhibitors of the ERK, PI3K-AKT, and Wnt signaling pathways mimics the effect of ATM deficiency on breast CAF proliferation. This is mainly ascribed to the β-catenin suppression and down-regulation of c-Myc, thus further leading to the decreased cyclinD1, cyclinE, and E2F1 expression and the enhanced p21Cip1 level. Our results reveal an important role of oxidized ATM in the regulation of the abnormal proliferation of breast CAFs. Oxidized ATM could serve as a potential target for treating breast cancer.

Therapeutic application of ultrasound-guided 8-gauge Mammotome system in presumed benign breast lesions.

Abstract:  The stereotactic or ultrasound‐guided vacuum‐assisted breast biopsy (Mammotome, MMT) system is a minimally invasive surgical technique. Increasingly, it is used to remove benign breast lesions as management. To evaluate the therapeutic value of 8‐gauge ultrasound‐guided MMT system (UMS) in presumed benign breast lesions, a retrospective analysis was performed on a series of 2,167 consecutive 8‐gauge UMS procedures. The parameters used in this analysis included lesion size, location, breast imaging reporting and data system for ultrasound category, histopathologic diagnosis, and others. A total of 1,119 women whose mean ages were 36.6 years (range: 12–71, SD: 9.6) underwent 2,167 consecutive 8‐gauge UMS procedures. Among the patients, 298 cases (26.63%) did not have palpable mass, 430 (38.43%) had multiple lesions, and 237 (21.18%) had bilateral ones. The average size of excised lesions was 15.8 mm (range: 5–55, SD: 6.7) in the largest dimension, including 294 lesions < 10 mm (13.57%); 1,359 lesions, 10–19 mm (62.71%); 420 lesions, 20–29 mm (19.38%), and 94 lesions ≥ 30 mm (4.34%). Predominant lesions (81.59%) were solid on ultrasound image and nearly half (48.59%) of them were localized in the upper outer quadrant. Histopathologic diagnosis revealed that the overwhelming majority of specimens (96.61%) were benign, most of which manifested as fibroadenoma and fibrocystic changes, while high‐risk lesions were revealed in 31 (2.29%) cases and malignancies in 15 (1.11%). Average time for procedure was 8.6 minutes (range: 3.5–38, SD: 5.4) and mean number of cores removed in the procedure was 9.3 (range: 2–42, SD: 3.7). Complete excision was achieved predominantly (99.82%). Complications (59, 5.27%) in which hematoma (41, 3.66%) was the majority were acceptable. In conclusion, the 8‐gauge UMS procedure is a safe and potent therapeutic management with satisfactory cosmetic outcome for benign and high‐risk breast lesions, especially for bilateral, multiple, and nonpalpable ones. It is a reliable biopsy tool for suspected lesions as well. Eradication and maximal cosmesis can be achieved with few complications if the targeted lesion is limited to 30 mm.

Nuclear Drosha enhances cell invasion via an EGFR-ERK1/2-MMP7 signaling pathway induced by dysregulated miRNA-622/197 and their targets LAMC2 and CD82 in gastric cancer

Drosha is an RNA III-like enzyme that has an aberrant expression in some tumors. Our previous studies showed the aberrant Drosha in gastric tumors. However, the roles of nuclear Drosha, the main regulator of microRNA (miRNA) biogenesis, in gastric cancer (GC) progression remain poorly understood. In this study, we demonstrated that nuclear Drosha is significantly associated with cell invasion of GC and that Drosha silence impedes the tumor invasion. Knockdown of Drosha led to a set of dysregulated miRNAs in GC cells. Multiple targets of these miRNAs were the members in cell migration, invasion and metastasis-associated signaling (e.g. ECM-receptor interaction, focal adhesion, p53 signaling and MAPK signaling pathway) revealed by bioinformatics analysis. LAMC2 (a key element of ECM-receptor signaling) and CD82 (a suppressor of p53 signaling) are the targets of miR-622 and miR-197, respectively. High levels of LAMC2 and low levels of CD82 were significantly related to the worse outcome for GC patients. Furthermore, overexpression of LAMC2 and knockdown of CD82 markedly promoted GC cell invasion and activated EGFR/ERK1/2-MMP7 signaling via upregulation of the expression of phosphorylated (p)-EGFR, p-ERK1/2 and MMP7. Our findings suggest that nuclear Drosha potentially has a role in the development of GC.