The risk variant rs11836367 contributes to breast cancer onset and metastasis by attenuating Wnt signaling via regulating NTN4 expression.

Most genome-wide association study (GWAS)-identified breast cancer-associated causal variants remain uncharacterized. To provide a framework of understanding GWAS-identified variants to function, we performed a comprehensive study of noncoding regulatory variants at the NTN4 locus (12q22) and NTN4 gene in breast cancer etiology. We find that rs11836367 is the more likely causal variant, disrupting enhancer activity in both enhancer reporter assays and endogenous genome editing experiments. The protective T allele of rs11837367 increases the binding of GATA3 to the distal enhancer and up-regulates NTN4 expression. In addition, we demonstrate that loss of NTN4 gene in mice leads to tumor earlier onset, progression, and metastasis. We discover that NTN4, as a tumor suppressor, can attenuate the Wnt signaling pathway by directly binding to Wnt ligands. Our findings bridge the gaps among breast cancer-associated single-nucleotide polymorphisms, transcriptional regulation of NTN4, and breast cancer biology, which provides previously unidentified insights into breast cancer prediction and prevention.

Extracellular ATP promotes breast cancer chemoresistance via HIF-1α signaling.

We have previously demonstrated that extracellular adenosine 5'-triphosphate (ATP) promotes breast cancer cell chemoresistance. However, the underlying mechanism remains unclear. Using a cDNA microarray, we demonstrated that extracellular ATP can stimulate hypoxia-inducible factor (HIF) signaling. In this study, we report that hypoxia-inducible factor 1α (HIF-1α) was upregulated after ATP treatment and mediated the ATP-driven chemoresistance process. We aimed to investigate the mechanisms and identify potential clinically relevant targets that are involved. Using mass spectrometry, we found that aldolase A (ALDOA) interacts with HIF-1α and increases HIF-1α expression. We then demonstrated that STAT3-ALDOA mediates ATP-HIF-1α signaling and upregulates the HIF-1 target genes adrenomedullin (ADM) and phosphoinositide-dependent kinase-1 (PDK1). Moreover, we show that PI3K/AKT acts upstream of HIF-1α in ATP signaling and contributes to chemoresistance in breast cancer cells. In addition, HIF-1α-knockdown or treatment with direct HIF inhibitors combined with the ATP hydrolase apyrase in MDA-MB-231 cells induced enhanced drug sensitivity in nude BALB/c mice. We then used in vitro spheroid formation assays to demonstrate the significance of ATP-HIF-1α in mediating chemoresistance. Furthermore, considering that indirect HIF inhibitors are effective in clinical cancer therapy, we treated tumor-bearing BALB/c mice with STAT3 and PI3K/AKT inhibitors and found that the dual-targeting strategy sensitized breast cancer to cisplatin. Finally, using breast cancer tissue microarrays, we found that ATP-HIF-1α signaling is associated with cancer progression, poor prognosis, and resistance to chemotherapy. Taken together, we suggest that HIF-1α signaling is vital in ATP-driven chemoresistance and may serve as a potential target for breast cancer therapies.

Extracellular ATP promotes breast cancer invasion and chemoresistance via SOX9 signaling.

Our previous research demonstrated that extracellular adenosine 5'-triphosphate (ATP) could promote breast cancer cell invasion. However, the impact of extracellular ATP on chemoresistance and the mechanisms behind ATP pro-invasion and pro-chemoresistance remain unclear. Here we aimed to determine the molecules or signaling pathways involved. cDNA microarray was performed to identify the differentially expressed genes before and after ATP treatment. As a result, Sex-determining region Y-box 9 (SOX9) was up-regulated after ATP treatment in breast cancer cells. In vitro invasion and migration assays demonstrated that knocking down SOX9 attenuated ATP-driven invasive capability. Mass spectrometry and co-IP revealed that SOX9 interacted with Janus kinase 1 (JAK1). Afterward, IL-6-JAK1-STAT3 signaling was demonstrated to promote SOX9 expression and invasion following ATP treatment. Notably, ATP-IL-6-SOX9 signaling was shown to stimulate chemoresistance in breast cancer cells. ChIP assays identified some potential SOX9 target genes, among which carcinoembryonic antigen-related cell adhesion molecule 5/6 (CEACAM5/6) was demonstrated to mediate ATP pro-invasive function, while ATP-binding cassette subfamily B member 1 (ABCB1) and ATP-binding cassette subfamily G member 2 (ABCG2) mediated ATP-driven chemoresistance. In addition, SOX9-knockdown and apyrase (an ATP hydrolase)-treated MDA-MB-231 cells illustrated decreased tumor growth and enhanced drug sensitivity in nude mice. In vitro spheroid formation assays also proved the significance of ATP-SOX9 in mediating chemoresistance. Moreover, molecules involved in ATP-SOX9 signaling were up-regulated in human breast carcinoma specimens and were associated with poor prognosis. Altogether, SOX9 signaling is vital in ATP-driven invasion and chemoresistance, which may serve as a potential target for breast cancer therapies.

Extracellular ATP promotes breast cancer invasion and epithelial-mesenchymal transition via hypoxia-inducible factor 2α signaling.

Extracellular ATP has been shown to play an important role in invasion and the epithelial-mesenchymal transition (EMT) process in breast cancer; however, the mechanism is unclear. Here, by using a cDNA microarray, we demonstrated that extracellular ATP could stimulate hypoxia-inducible factor (HIF) signaling and upregulate hypoxia-inducible factor 1/2α (HIF-1/2α) expression. After knocking down HIF-1/2α using siRNA, we found that ATP-driven invasion and EMT were significantly attenuated via HIF2A-siRNA in breast cancer cells. By using ChIP assays, we revealed that the biological function of extracellular ATP in invasion and EMT process depended on HIF-2α direct targets, among which lysyl oxidase-like 2 (LOXL2) and matrix metalloproteinase-9 (MMP-9) mediated ATP-driven invasion, and E-cadherin and Snail mediated ATP-driven EMT, respectively. In addition, using silver staining and mass spectrometry, we found that phosphoglycerate kinase 1 (PGK1) could interact with HIF-2α and mediate ATP-driven HIF-2α upregulation. Furthermore, we demonstrated that expressions of HIF-2α and its target proteins could be regulated via ATP by AKT-PGK1 pathway. Using a Balb/c mice model, we illustrated the function of HIF-2α in promoting tumor growth and metastasis in vivo. Moreover, by exploring online databases, we found that molecules involved in ATP-HIF-2α signaling were highly expressed in human breast carcinoma tissues and were associated with poor prognosis. Altogether, these findings suggest that extracellular ATP could promote breast carcinoma invasion and EMT via HIF-2α signaling, which may be a potential target for future anti-metastasis therapy.

Genetic polymorphisms in CDH1 are associated with endometrial carcinoma susceptibility among Chinese Han women.

The cadherin 1 (CDH1) gene plays critical roles in the epithelial-mesenchymal transition process, potentially offering us a glimpse into the development of endometrial carcinoma (EC). The present study aimed to identify whether genetic variants in CDH1 affect EC susceptibility in Chinese Han women, using a strategy combining haplotype-tagging single nucleotide polymorphisms (htSNPs) association analysis with fine-scale mapping. A total of 9 htSNPs in CDH1 were genotyped among 516 cases and 706 age-matched cancer-free controls. Logistic regression analyses revealed 3 htSNPs (rs17715799, rs6499199 and rs13689) to be associated with increased EC risk and 3 htSNPs (rs12185157, rs10431923 and rs4783689) with decreased EC risk. Furthermore, 14 newly imputed SNPs of CDH1 were identified to be associated with EC risk (P<0.05) using genotype imputation analysis. Notably, multivariate logistic analysis demonstrated that rs13689, rs10431923 and rs10431924 could affect EC susceptibility independently (P≤0.001). Subsequent Generalized Multifactor Dimensionality Reduction analysis revealed several best fitting models for predicting EC risk, including SNP-SNP interactions among rs7100190, rs12185157, rs10431923, rs7186053, rs6499199, rs4783689, rs13689, rs6499197 and rs10431924, and SNP-environment interactions between related SNPs and number of childbirth. Moreover, functional annotations suggest that the majority of these susceptible variants may carry potential biological functions that affect certain gene regulatory elements. In summary, this study suggested that the genetic polymorphisms of CDH1 were indeed associated with EC susceptibility on several levels. If further additional functional studies could verify these findings, these genetic variants may serve as future personalized markers for the early prediction of endometrial cancer in Chinese Han women.

Associations of gene­wide SNPs in SNAI1 and TWIST1 with breast cancer and ovarian cancer susceptibility among Chinese Han women.

Extensive evidence suggests that the genetic etiologies of breast cancer (BC) and ovarian cancer (OC) show a certain degree of similarity. This study aimed to find out whether the single nucleotide polymorphisms (SNPs) of genes SNAI1 and TWIST1 may affect BC and OC susceptibility. A total of 7 tagging­SNPs (tSNPs) were directly genotyped in 1,161 BC cases, 286 OC cases and 1,273 cancer­free controls among Chinese Han women. Twenty­eight variants in these 2 genes were genotyped by 'in silico' genotype imputation. Logistic regression (LR) revealed that tSNPs SNAI1 rs6125849, TWIST1 rs4721746 and TWIST1 rs4721745 were protective genetic variants for BC/OC. Allelic association tests of gene­wide SNPs demonstrated that the minor alleles of SNAI1 rs6125849, TWIST1 rs4721745 and TWIST1 rs11973396 were strongly associated with BC/OC susceptibility. Multivariate LR presented that SNAI1 rs6125849, TWIST1 rs4721745, rs4721746 and rs11973396 affected BC/OC susceptibility independently, and women harboring all four protective genoytpes had the lowest risk. Multifactor dimensionality reduction analysis further showed that SNAI1 rs6125849 and TWIST1 rs4721745 had the strongest synergistic interaction. Functional annotation predicted that the minor alleles of SNAI1 rs6125849 and TWIST1 rs4721745 altered their binding affinities with transcription factors E2F6 and TCF11­MafG respectively. These results indicate that genetic variants in SNAI1 and TWIST1, most probably SNAI1 rs6125849 and TWIST1 rs4721745, may modulate BC and OC susceptibility.

Extracellular ATP drives breast cancer cell migration and metastasis via S100A4 production by cancer cells and fibroblasts.

Our previous work has demonstrated that extracellular ATP is an important pro-invasive factor, and in this study, we tapped into a possible mechanism involved. We discovered that ATP could upregulate both the intracellular expression and secretion of S100A4 in breast cancer cells and fibroblasts. Apart from stimulating breast cancer cell motility via intracellular S100A4, ATP enhanced the ability of breast cancer cells to transform fibroblasts into cancer-associated fibroblast (CAF)-like cells, which in turn secreted S100A4 to further promote cancer cell motility. Both apyrase and niclosamide treatments could inhibit metastasis of inoculated tumors to lung, liver and kidney in mice model, and CAFs from these treated tumors exhibited weakened migration-stimulating capacity for breast cancer cells. Collectively, our data indicate that extracellular ATP promotes the interactions between breast cancer cells and fibroblasts, which work collaboratively via production of S100A4 to exacerbate breast cancer metastasis.

Diagnosis and Differential Diagnosis of Desmoplastic Fibroblastoma by Clinical, Radiological, and Histopathological Analyses.

BACKGROUND: Desmoplastic fibroblastoma (collagenous fibroma) is an uncommon benign soft-tissue tumor, rarely involving bone. It shares some overlapping features with other infiltrate tumors, such as desmoid-type fibromatosis, neurofibroma, and low-grade fibromyxoid sarcoma. The misdiagnosis may cause unnecessary surgical overtreatment, especially for those involving bone. In order to deepen the understanding of the diagnosis and differential diagnosis of desmoplastic fibroblastoma, we planned to analyze the clinical, radiological, and histopathological features and the outcome of desmoplastic fibroblastoma on the basis of case analysis and literature review. METHODS: Sixteen cases were retrieved from the surgical pathology records from May 2011 to April 2016 in the Department of Pathology in Beijing Jishuitan Hospital. Formalin-fixed, paraffin-embedded specimens of 16 cases of desmoplastic fibroblastoma were collected. Hematoxylin and eosin stain and immunohistochemistry were used to observe the histological features of desmoplastic fibroblastoma of soft tissue and bone. The images for diagnosis obtained from the ultrasonic examination, X-ray, magnetic resonance imaging, and computed tomography were used to observe the radiological features. Related literatures were retrieved from the PubMed and CNKI databases. RESULTS: Sixteen cases of desmoplastic fibroblastoma of soft tissue were located in the hand (n = 7), foot (n = 4), upper arm (n = 1), shoulder (n = 1), forearm (n = 2), and one case occurred in the proximal femur. Age ranged from 32 to 82 years (median age: 58 years). There were six females and ten males. Histologically, the lesions of soft tissue appeared as well-circumscribed masses with abundant collagenous matrix and low vascularity. Tumor cells were stellate- or spindle-shaped and uniformly distributed within the extracellular matrix. In five cases, the desmoplastic fibroblastoma were found to have infiltrated into the skeletal muscle tissue. In one case of desmoplastic fibroblastoma of bone, radiographs revealed osteolytically well-defined lesion. Immunohistochemistry stain showed that vimentin and smooth muscle actin were positive in all cases of desmoplastic fibroblastoma. CONCLUSIONS: Desmoplastic fibroblastoma (collagenous fibroma) has prominent clinical, histopathological, and radiological features. Before the differential diagnosis from other tumors is obtained by thorough analysis and comparison of the similar and different characteristics, the appropriate surgical management and accurate prognosis evaluation could not be delivered to the patient.