Yate-Ching Yuan

FZD7 has a critical role in cell proliferation in triple negative breast cancer

Breast cancer is genetically and clinically heterogeneous. Triple negative breast cancer (TNBC) is a subtype of breast cancer that is usually associated with poor outcome and lack of benefit from targeted therapy. We used microarray analysis to perform a pathway analysis of TNBC compared with non-triple negative breast cancer (non-TNBC). Overexpression of several Wnt pathway genes, such as frizzled homolog 7 (FZD7), low density lipoprotein receptor-related protein 6 and transcription factor 7 (TCF7) was observed in TNBC, and we directed our focus to the Wnt pathway receptor, FZD7. To validate the function of FZD7, FZD7shRNA was used to knock down FZD7 expression. Notably, reduced cell proliferation and suppressed invasiveness and colony formation were observed in TNBC MDA-MB-231 and BT-20 cells. Study of the possible mechanism indicated that these effects occurred through silencing of the canonical Wnt signaling pathway, as evidenced by loss of nuclear accumulation of β-catenin and decreased transcriptional activity of TCF7. In vivo studies revealed that FZD7shRNA significantly suppressed tumor formation, through reduced cell proliferation, in mice bearing xenografts without FZD7 expression. Our findings suggest that FZD7-involved canonical Wnt signaling pathway is essential for tumorigenesis of TNBC, and thus, FZD7 shows promise as a biomarker and a potential therapeutic target for TNBC.

MicroRNA profiling of clear cell renal cell carcinoma by whole-genome small RNA deep sequencing of paired frozen and formalin-fixed, paraffin-embedded tissue specimens.

Renal cell carcinoma (RCC) is one of the leading causes of cancer mortality. Characterization of microRNA (miRNA) expression of RCC will help disclose new pathogenic pathways in tumourigenesis and progression and may lead to the development of molecular biomarkers and target‐specific therapies for diagnosis, prognostication and treatment. With limitations in test specificity and the ability to detect novel miRNA and other small non‐coding RNAs (smRNAs), microarray and RT–PCR techniques are being replaced by the evolving deep‐sequencing technologies, at least in the discovery phase. Until now, cancer miRNA profiling of human benign and tumour specimen sets, using smRNA deep‐sequencing (smRNA‐seq), has not been reported. Specifically, due to concern over possible poor RNA quality/integrity, formalin‐fixed paraffin‐embedded (FFPE) samples have not been used for such studies. Here, we performed whole‐genome smRNA‐seq analysis using a benign and RCC specimen set and have successfully profiled the miRNA expression. Studies performed on paired frozen and FFPE specimens showed very similar results. Moreover, a comparison study of microarray, deep‐sequencing and RT–PCR methodologies also showed a high correlation among the three technologies. To our knowledge, this is the first study to demonstrate that FFPE specimens can be used reliably for miRNA deep‐sequencing analysis, making future large‐scale clinical cohort/trial‐based studies possible. Copyright

De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer

BackgroundMicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome.MethodsThe pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers.ResultsMore than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients.ConclusionsOur study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.

Profiles of epigenetic histone post-translational modifications at type 1 diabetes susceptible genes

Background: Both genetic and epigenetic factors are implicated in Type 1 diabetes (T1D). Results: Variations in histone H3-lysine 9 acetylation are detected around the promoter/enhancer regions of key T1D susceptible genes in monocytes of T1D subjects versus normals. Conclusion: The chromatin status of this key region is altered in T1D. Significance: Epigenetic variations at T1D susceptible genes may be functionally important. Both genetic and environmental factors are implicated in type 1 diabetes (T1D). Because environmental factors can trigger epigenetic changes, we hypothesized that variations in histone post-translational modifications (PTMs) at the promoter/enhancer regions of T1D susceptible genes may be associated with T1D. We therefore evaluated histone PTM variations at known T1D susceptible genes in blood cells from T1D patients versus healthy nondiabetic controls, and explored their connections to T1D. We used the chromatin immunoprecipitation-linked to microarray approach to profile key histone PTMs, including H3-lysine 4 trimethylation (H3K4me3), H3K27me3, H3K9me3, H3K9 acetylation (H3K9Ac), and H4K16Ac at genes within the T1D susceptible loci in lymphocytes, and H3K4me3, H3K9me2, H3K9Ac, and H4K16Ac at the insulin-dependent diabetes mellitus 1 region in monocytes of T1D patients and healthy controls separately. We screened for potential variations in histone PTMs using computational methods to compare datasets from T1D and controls. Interestingly, we observed marked variations in H3K9Ac levels at the upstream regions of HLA-DRB1 and HLA-DQB1 within the insulin-dependent diabetes mellitus 1 locus in T1D monocytes relative to controls. Additional experiments with THP-1 monocytes demonstrated increased expression of HLA-DRB1 and HLA-DQB1 in response to interferon-γ and TNF-α treatment that were accompanied by changes in H3K9Ac at the same promoter regions as that seen in the patient monocytes. These results suggest that the H3K9Ac status of HLA-DRB1 and HLA-DQB1, two genes highly associated with T1D, may be relevant to their regulation and transcriptional response toward external stimuli. Thus, the promoter/enhancer architecture and chromatin status of key susceptible loci could be important determinants in their functional association to T1D susceptibility.

The FEN1 E359K germline mutation disrupts the FEN1–WRN interaction and FEN1 GEN activity, causing aneuploidy-associated cancers

Polymorphisms and somatic mutations in Flap Endonuclease 1 (FEN1), an essential enzyme involved in DNA replication and repair, can lead to functional deficiencies of the FEN1 protein and a predisposition to cancer. We identified a FEN1 germline mutation that changed residue E359 to K in a patient whose family had a history of breast cancer. We determined that the E359K mutation, which is in the protein–protein domain of FEN1, abolished the interaction of FEN1 with Werner syndrome protein (WRN), an interaction that is critical for resolving stalled DNA replication forks. Furthermore, although the flap endonuclease activity of FEN1 E359K was unaffected, it failed to resolve bubble structures, which require the FEN1 gap-dependent endonuclease activity. To determine the etiological significance of E359K, we established a mouse model containing this mutation. E359K mouse embryonic fibroblasts (MEF) were more sensitive to DNA crosslinking agents that cause replication forks to stall. Cytological analysis suggested that the FEN1–WRN interaction was also required for telomere stability; mutant cell lines had fragile telomeres, increased numbers of spontaneous chromosomal anomalies and higher frequencies of transformation. Moreover, the incidence of cancer was significantly higher in mice homozygous for FEN1 E359K than in wild-type mice, suggesting that the FEN1 E359K mutation is oncogenic.

SERPINA1 is a direct estrogen receptor target gene and a predictor of survival in breast cancer patients.

Of all breast cancer patients, about 70% are ER+ and 10% are ER+/HER2+. The ER+/HER2+ patients have a worse outcome compared to ER+/HER2- patients. Currently there is a lack of effective prognosis biomarkers for the prediction of outcome in ER+/HER2+ patients. Genome-wide differences in ER binding between the endocrine-responsive and endocrine-resistant cells were discovered using ChIP-seq, and combined with gene expression microarray data to identify direct ER target genes. These genes were correlated to survival outcome using publicly available breast cancer patient cohorts. We found the expression of the gene SERPINA1 to have a significant predictive value for the overall survival (OS) of ER+ patients in the TCGA cohort, and validated this finding in the Curtis cohort. SERPINA1 also has a significant predictive value for the OS of ER+/HER2+ patients in the TCGA cohort, with validation in the Bild cohort. The expression of SERPINA1 can be suppressed by fulvestrant and HER2 siRNA. Our results indicate that ER is constitutively activated, resulting in an E2-independent ER binding to the SERPINA1 gene and upregulation of SERPINA1 expression. Importantly, results of survival correlation suggests that high expression of SERPINA1 could be predictive for a better clinical outcome of ER+ and ER+/HER2+ patients.

Genomic mutation-driven metastatic breast cancer therapy: a single center experience

Background Next-Generation Sequencing (NGS) has made genomic mutation-driven therapy feasible for metastatic breast cancer (MBC) patients. We frequently submit tumor tissue from MBC patients for targeted NGS of tumor using the Illumina HiSeq 2000 platform (FoundationOne®, Foundation Medicine, MA). Herein, we report the results and clinical impact of this test in MBC patients. Patients and Methods We identified patients with MBC treated at City of Hope from January 2014 to May 2016 who underwent NGS. Patients’ clinical characteristics, response to treatment (clinical assessment of tumor regression), and genomic mutation profiles were reviewed. Results Forty-four patients with MBC underwent NGS: 24 triple negative breast cancer, 16 estrogen receptor positive, and 4 human epidermal growth factor receptor 2 positive patients. Twenty-three patients received more than three lines of chemotherapy prior to NGS. Actionable mutations (potentially responsive to targeted therapies that are on the market or in registered clinical trials) were identified in almost all patients (42/44; 95%) and over half of these 42 patients with actionable mutations (23/42; 55%) initiated mutation-driven targeted therapies. Of these 23 patients, 16/23 (70%) had assessable responses, and 7/23 (30%) were not assessable for response due to short exposure (<2 weeks) or hospice transition. The remaining 19/42 (45%) patients did not initiate targeted therapy. Conclusion NGS can identify effective targeted therapy options for MBC patients based on actionable mutations that were not previously offered based on pathology type. NGS should be performed early in patients with good performance status and preferably in clinical settings where genomic mutation-driven therapeutic trials are available.

Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

Deregulated monoubiquitination of histone H2B (H2Bub1), mainly catalyzed by E3 ubiquitin‐protein ligase RNF20/RNF40 complex, may play an important role in cancer. Here we investigate potential roles of H2Bub1 and the underlying mechanisms through which it contributes to cancer development and progression in lung adenocarcinoma. We show that downregulation of H2Bub1 through RNF20 knockdown dramatically decreases H3K79 and H3K4 trimethylation in both normal and malignant lung epithelial cell lines. Concurrently, global transcriptional profiling analysis reveals that multiple tumor‐associated genes such as CCND3, E2F1/2, HOXA1, Bcl2 modifying factor (BMF), Met, and Myc; and signaling pathways of cellular dedifferentiation, proliferation, adhesion, survival including p53, cadherin, Myc, and anti‐apoptotic pathways are differentially expressed or significantly altered in these lung epithelial cells upon downregulation of H2Bub1. Moreover, RNF20 knockdown dramatically suppresses terminal squamous differentiation of cultured bronchial epithelial cells, and significantly enhances proliferation, migration, invasion, and cisplatin resistance of lung cancer cells. Furthermore, immunohistochemistry analysis shows that H2Bub1 is extremely low or undetectable in >70% of 170 lung adenocarcinoma samples. Notably, statistical analysis demonstrates that loss of H2Bub1 is significantly correlated with poor differentiation in lung adenocarcinoma (p = 0.0134). In addition, patients with H2Bub1‐negative cancers had a trend towards shorter survival compared with patients with H2Bub1‐positive cancers. Taken together, our findings suggest that loss of H2Bub1 may enhance malignancy and promote disease progression in lung adenocarcinoma probably through modulating multiple cancer signaling pathways.

Identification of core aberrantly expressed microRNAs in serous ovarian carcinoma

MicroRNAs (miRNAs) have recently demonstrated great potential and enormous promise in the diagnosis, prognosis and therapy of various types of cancer. In this study, we performed a comprehensive miRNA expression analysis in the omental metastasis of serous ovarian carcinoma (SOC) using small RNA sequencing. Two hundred and fifty-one aberrantly expressed miRNAs were identified, which clearly separated malignant omentum from normal omentum. Furthermore, miRNA profiles in primary chemo-sensitive and chemo-resistant/refractory SOC were determined using publicly available data. Comparing miRNA expression profiles in omental metastases and primary chemo-sensitive and chemo-resistant/refractory tumors, a set of 70 miRNAs that were aberrantly expressed in both primary and metastatic SOC has been identified for the first time. These core aberrantly expressed miRNAs may play crucial roles in the tumorigenesis, growth, and metastasis of SOC. Therefore, they can serve as potential diagnostic biomarkers and as therapeutic targets for miRNA-mediated therapy. Kaplan–Meier overall survival analysis using The Cancer Genome Atlas data demonstrated that 10 miRNAs (hsa-miR-135, 150, -340, 625, 1908, 3187, -96, -196b, -449c, and -1275) were associated with survival of patients with SOC, which may serve as potential prognostic biomarkers.

Targeting histone methyltransferase G9a inhibits growth and Wnt signaling pathway by epigenetically regulating HP1α and APC2 gene expression in non-small cell lung cancer

BackgroundDysregulated histone methyltransferase G9a may represent a potential cancer therapeutic target. The roles of G9a in tumorigenesis and therapeutics are not well understood in non-small cell lung cancer (NSCLC). Here we investigated the impact of G9a on tumor growth and signaling pathways in NSCLC.MethodsImmunohistochemistry analyzed G9a expression in NSCLC tissues. Both siRNA and selective inhibitor were used to target G9a. The impact of targeting G9a on key genes, signaling pathways and growth were investigated in NSCLC cells by RNA sequencing analysis, rescue experiments, and xenograft models.ResultsOverexpression of G9a (≥ 5% of cancer cells showing positive staining) was found in 43.2% of 213 NSCLC tissues. Multiple tumor-associated genes including HP1α, APC2 are differentially expressed; and signaling pathways involved in cellular growth, adhesion, angiogenesis, hypoxia, apoptosis, and canonical Wnt signaling pathways are significantly altered in A549, H1299, and H1975 cells upon G9a knockdown. Additionally, targeting G9a by siRNA-mediated knockdown or by a selective G9a inhibitor UNC0638 significantly inhibited tumor growth, and dramatically suppressed Wnt signaling pathway in vitro and in vivo. Furthermore, we showed that treatment with UNC0638 restores the expression of APC2 expression in these cells through promoter demethylation. Restoring HP1α and silencing APC2 respectively attenuated the inhibitory effects on cell proliferation and Wnt signaling pathway in cancer cells in which G9a was silenced or suppressed.ConclusionsThese findings demonstrate that overexpressed G9a represents a promising therapeutic target, and targeting G9a potentially suppresses growth and Wnt signaling pathway partially through down-regulating HP1α and epigenetically restoring these tumor suppressors such as APC2 that are silenced in NSCLC.