Soonweng Cho

Novel Methylated Biomarkers and a Robust Assay to Detect Circulating Tumor DNA in Metastatic Breast Cancer

The ability to consistently detect cell-free tumor-specific DNA in peripheral blood of patients with metastatic breast cancer provides the opportunity to detect changes in tumor burden and to monitor response to treatment. We developed cMethDNA, a quantitative multiplexed methylation-specific PCR assay for a panel of ten genes, consisting of novel and known breast cancer hypermethylated markers identified by mining our previously reported study of DNA methylation patterns in breast tissue (103 cancer, 21 normal on the Illumina HumanMethylation27 Beadchip) and then validating the 10-gene panel in The Cancer Genome Atlas project breast cancer methylome database. For cMethDNA, a fixed physiologic level (50 copies) of artificially constructed, standard nonhuman reference DNA specific for each gene is introduced in a constant volume of serum (300 μL) before purification of the DNA, facilitating a sensitive, specific, robust, and quantitative assay of tumor DNA, with broad dynamic range. Cancer-specific methylated DNA was detected in training (28 normal, 24 cancer) and test (27 normal, 33 cancer) sets of recurrent stage IV patient sera with a sensitivity of 91% and a specificity of 96% in the test set. In a pilot study, cMethDNA assay faithfully reflected patient response to chemotherapy (N = 29). A core methylation signature present in the primary breast cancer was retained in serum and metastatic tissues collected at autopsy two to 11 years after diagnosis of the disease. Together, our data suggest that the cMethDNA assay can detect advanced breast cancer, and monitor tumor burden and treatment response in women with metastatic breast cancer.

HOXB13 mediates tamoxifen resistance and invasiveness in human breast cancer by suppressing ERα and inducing IL-6 expression

Most breast cancers expressing the estrogen receptor α (ERα) are treated successfully with the receptor antagonist tamoxifen (TAM), but many of these tumors recur. Elevated expression of the homeodomain transcription factor HOXB13 correlates with TAM-resistance in ERα-positive (ER+) breast cancer, but little is known regarding the underlying mechanism. Our comprehensive evaluation of HOX gene expression using tiling microarrays, with validation, showed that distant metastases from TAM-resistant patients also displayed high HOXB13 expression, suggesting a role for HOXB13 in tumor dissemination and survival. Here we show that HOXB13 confers TAM resistance by directly downregulating ERα transcription and protein expression. HOXB13 elevation promoted cell proliferation in vitro and growth of tumor xenografts in vivo. Mechanistic investigations showed that HOXB13 transcriptionally upregulated interleukin (IL)-6, activating the mTOR pathway via STAT3 phosphorylation to promote cell proliferation and fibroblast recruitment. Accordingly, mTOR inhibition suppressed fibroblast recruitment and proliferation of HOXB13-expressing ER+ breast cancer cells and tumor xenografts, alone or in combination with TAM. Taken together, our results establish a function for HOXB13 in TAM resistance through direct suppression of ERα and they identify the IL-6 pathways as mediator of disease progression and recurrence.

Association of BRAFV600E Mutation and MicroRNA Expression with Central Lymph Node Metastases in Papillary Thyroid Cancer: A Prospective Study from Four Endocrine Surgery Centers

BACKGROUND Studies have demonstrated an association of the BRAF(V600E) mutation and microRNA (miR) expression with aggressive clinicopathologic features in papillary thyroid cancer (PTC). Analysis of BRAF(V600E) mutations with miR expression data may improve perioperative decision making for patients with PTC, specifically in identifying patients harboring central lymph node metastases (CLNM). METHODS Between January 2012 and June 2013, 237 consecutive patients underwent total thyroidectomy and prophylactic central lymph node dissection (CLND) at four endocrine surgery centers. All tumors were tested for the presence of the BRAF(V600E) mutation and miR-21, miR-146b-3p, miR-146b-5p, miR-204, miR-221, miR-222, and miR-375 expression. Bivariate and multivariable analyses were performed to examine associations between molecular markers and aggressive clinicopathologic features of PTC. RESULTS Multivariable logistic regression analysis of all clinicopathologic features found miR-146b-3p and miR-146b-5p to be independent predictors of CLNM, while the presence of BRAF(V600E) almost reached significance. Multivariable logistic regression analysis limited to only predictors available preoperatively (molecular markers, age, sex, and tumor size) found miR-146b-3p, miR-146b-5p, miR-222, and BRAF(V600E) mutation to predict CLNM independently. While BRAF(V600E) was found to be associated with CLNM (48% mutated in node-positive cases vs. 28% mutated in node-negative cases), its positive and negative predictive values (48% and 72%, respectively) limit its clinical utility as a stand-alone marker. In the subgroup analysis focusing on only classical variant of PTC cases (CVPTC), undergoing prophylactic lymph node dissection, multivariable logistic regression analysis found only miR-146b-5p and miR-222 to be independent predictors of CLNM, while BRAF(V600E) was not significantly associated with CLNM. CONCLUSION In the patients undergoing prophylactic CLNDs, miR-146b-3p, miR-146b-5p, and miR-222 were found to be predictive of CLNM preoperatively. However, there was significant overlap in expression of these miRs in the two outcome groups. The BRAF(V600E) mutation, while being a marker of CLNM when considering only preoperative variables among all histological subtypes, is likely not a useful stand-alone marker clinically because the difference between node-positive and node-negative cases was small. Furthermore, it lost significance when examining only CVPTC. Overall, our results speak to the concept and interpretation of statistical significance versus actual applicability of molecular markers, raising questions about their clinical usefulness as individual prognostic markers.

The Notch Pathway Inhibits TGFβ Signaling in Breast Cancer through HEYL-Mediated Crosstalk

Acquired resistance to TGFβ is a key step in the early stages of tumorigenesis. Mutations in TGFβ signaling components are rare, and little is known about the development of resistance in breast cancer. On the other hand, an activated Notch pathway is known to play a substantial role in promoting breast cancer development. Here, we present evidence of crosstalk between these two pathways through HEYL. HEYL, a basic helix-loop-helix transcription factor and a direct target of Notch signaling, is specifically overexpressed in breast cancer. HEYL represses TGFβ activity by binding to TGFβ-activated Smads. HeyL(-/-) mice have defective mammary gland development with fewer terminal end buds. On the other hand, HeyL transgenic mice show accelerated mammary gland epithelial proliferation and 24% of multiparous mice develop mammary gland cancer. Therefore, repression of TGFβ signaling by Notch acting through HEYL may promote initiation of breast cancer.

Subtype-specific overexpression of the Rac-GEF P-REX1 in breast cancer is associated with promoter hypomethylation

IntroductionThe Rac-GEF P-REX1 is a key mediator of ErbB signaling in breast cancer recently implicated in mammary tumorigenesis and metastatic dissemination. Although P-REX1 is essentially undetectable in normal human mammary epithelial tissue, this Rac-GEF is markedly upregulated in human breast carcinomas, particularly of the luminal subtype. The mechanisms underlying P-REX1 upregulation in breast cancer are unknown. Toward the goal of dissecting the mechanistic basis of P-REX1 overexpression in breast cancer, in this study we focused on the analysis of methylation of the PREX1 gene promoter.MethodsTo determine the methylation status of the PREX1 promoter region, we used bisulfite genomic sequencing and pyrosequencing approaches. Re-expression studies in cell lines were carried out by treatment of breast cancer cells with the demethylating agent 5-aza-2′-deoxycitidine. PREX1 gene methylation in different human breast cancer subtypes was analyzed from the TCGA database.ResultsWe found that the human PREX1 gene promoter has a CpG island located between -1.2 kb and +1.4 kb, and that DNA methylation in this region inversely correlates with P-REX1 expression in human breast cancer cell lines. A comprehensive analysis of human breast cancer cell lines and tumors revealed significant hypomethylation of the PREX1 promoter in ER-positive, luminal subtype, whereas hypermethylation occurs in basal-like breast cancer. Treatment of normal MCF-10A or basal-like cancer cells, MDA-MB-231 with the demethylating agent 5-aza-2′-deoxycitidine in combination with the histone deacetylase inhibitor trichostatin A restores P-REX1 levels to those observed in luminal breast cancer cell lines, suggesting that aberrant expression of P-REX1 in luminal breast cancer is a consequence of PREX1 promoter demethylation. Unlike PREX1, the pro-metastatic Rho/Rac-GEF, VAV3, is not regulated by methylation. Notably, PREX1 gene promoter hypomethylation is a prognostic marker of poor patient survival.ConclusionsOur study identified for the first time gene promoter hypomethylation as a distinctive subtype-specific mechanism for controlling the expression of a key regulator of Rac-mediated motility and metastasis in breast cancer.

Combined treatment with epigenetic, differentiating, and chemotherapeutic agents cooperatively targets tumor-initiating cells in triple-negative breast cancer

Efforts to induce the differentiation of cancer stem cells through treatment with all-trans retinoic acid (ATRA) have yielded limited success, partially due to the epigenetic silencing of the retinoic acid receptor (RAR)-β The histone deacetylase inhibitor entinostat is emerging as a promising antitumor agent when added to the standard-of-care treatment for breast cancer. However, the combination of epigenetic, cellular differentiation, and chemotherapeutic approaches against triple-negative breast cancer (TNBC) has not been investigated. In this study, we found that combined treatment of TNBC xenografts with entinostat, ATRA, and doxorubicin (EAD) resulted in significant tumor regression and restoration of epigenetically silenced RAR-β expression. Entinostat and doxorubicin treatment inhibited topoisomerase II-β (TopoII-β) and relieved TopoII-β-mediated transcriptional silencing of RAR-β Notably, EAD was the most effective combination in inducing differentiation of breast tumor-initiating cells in vivo Furthermore, gene expression analysis revealed that the epithelium-specific ETS transcription factor-1 (ESE-1 or ELF3), known to regulate proliferation and differentiation, enhanced cell differentiation in response to EAD triple therapy. Finally, we demonstrate that patient-derived metastatic cells also responded to treatment with EAD. Collectively, our findings strongly suggest that entinostat potentiates doxorubicin-mediated cytotoxicity and retinoid-driven differentiation to achieve significant tumor regression in TNBC. Cancer Res; 76(7); 2013-24. ©2016 AACR.

Optimizing the Use of Gene Expression Profiling in Early-Stage Breast Cancer

Purpose Gene expression profiling assays are frequently used to guide adjuvant chemotherapy decisions in hormone receptor-positive, lymph node-negative breast cancer. We hypothesized that the clinical value of these new tools would be more fully realized when appropriately integrated with high-quality clinicopathologic data. Hence, we developed a model that uses routine pathologic parameters to estimate Oncotype DX recurrence score (ODX RS) and independently tested its ability to predict ODX RS in clinical samples. Patients and Methods We retrospectively reviewed ordered ODX RS and pathology reports from five institutions (n = 1,113) between 2006 and 2013. We used locally performed histopathologic markers (estrogen receptor, progesterone receptor, Ki-67, human epidermal growth factor receptor 2, and Elston grade) to develop models that predict RS-based risk categories. Ordering patterns at one site were evaluated under an integrated decision-making model incorporating clinical treatment guidelines, immunohistochemistry markers, and ODX. Final locked models were independently tested (n = 472). Results Distribution of RS was similar across sites and to reported clinical practice experience and stable over time. Histopathologic markers alone determined risk category with > 95% confidence in > 55% (616 of 1,113) of cases. Application of the integrated decision model to one site indicated that the frequency of testing would not have changed overall, although ordering patterns would have changed substantially with less testing of estimated clinical risk-high or clinical risk-low cases and more testing of clinical risk-intermediate cases. In the validation set, the model correctly predicted risk category in 52.5% (248 of 472). Conclusion The proposed model accurately predicts high- and low-risk RS categories (> 25 or ≤ 25) in a majority of cases. Integrating histopathologic and molecular information into the decision-making process allows refocusing the use of new molecular tools to cases with uncertain risk.

Inhibitors of STAT3, β‐catenin, and IGF‐1R sensitize mouse PIK3CA‐mutant breast cancer to PI3K inhibitors

Although mutations in the phosphoinositide 3‐kinase catalytic subunit (PIK3CA) are common in breast cancer, PI3K inhibitors alone have shown modest efficacy. We sought to identify additional pathways altered in PIK3CA‐mutant tumors that might be targeted in combination with PI3K inhibitors. We generated two transgenic mouse models expressing the human PIK3CA‐H1047R‐ and the ‐E545K hotspot‐mutant genes in the mammary gland and evaluated their effects on development and tumor formation. Molecular analysis identified pathways altered in these mutant tumors, which were also targeted in multiple cell lines derived from the PIK3CA tumors. Finally, public databases were analyzed to determine whether novel pathways identified in the mouse tumors were altered in human tumors harboring mutant PIK3CA. Mutant mice showed increased branching and delayed involution of the mammary gland compared to parental FVB/N mice. Mammary tumors arose in 30% of the MMTV‐PIK3CA‐H1047R and in 13% of ‐E545K mice. Compared to MMTV‐Her‐2 transgenic mouse mammary tumors, H1047R tumors showed increased upregulation of Wnt/β‐catenin/Axin2, hepatocyte growth factor (Hgf)/Stat3, insulin‐like growth factor 2 (Igf‐2), and Igf‐1R pathways. Inhibitors of STAT3, β‐catenin, and IGF‐1R sensitized H1047R‐derived mouse tumor cells and PIK3CA‐H1047R overexpressing human HS578T breast cancer cells to the cytotoxic effects of PI3K inhibitors. Analysis of The Cancer Genome Atlas database showed that, unlike primary PIK3CA‐wild‐type and HER‐2+ breast carcinomas, PIK3CA‐mutant tumors display increased expression of AXIN2, HGF, STAT3, IGF‐1, and IGF‐2 mRNA and activation of AKT, IGF1‐MTOR, and WNT canonical signaling pathways. Drugs targeting additional pathways that are altered in PIK3CA‐mutant tumors may improve treatment regimens using PI3K inhibitors alone.

Abstract 3370: Comparative analyses of multi-omics profiles reveal distinctive molecular signatures of young Asian breast cancers

Breast cancers (BC) in younger, premenopausal patients (YBC) tend to be more aggressive with worse prognosis, higher chance of relapse and poorer response to endocrine therapies compared to breast cancers in older patients. The proportion of YBC (age ≤ 40) among BC in East Asia is estimated to be 16-32%, significantly higher than the 7% reported in Western countries. In addition, approximately half of the Asian BC patients were premenopausal compared to 15-30% in the West. To characterize the molecular bases of Asian YBC, we have performed whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) on tumor and matched normal samples from 168 Korean BC patients consisting of 106 YBC cases (age ≤ 40) and 62 OBC cases (age > 40). We then performed comparison analyses with the TCGA BC cohort consisting of 1,116 tumors from primarily Caucasian patients, also grouped by age into YBC (age ≤ 40), IBC (40 60). We performed logistic regression analyses to identify differentially expressed (DE) genes and pathways among age-based cohorts while controlling for the confounding effects of molecular subtype, tumor purity and stage. Within the Asian cohort, we found that estrogen response, endocrine therapy resistance, and various metabolism pathways are up-regulated in YBCs while cell cycle, proliferation and inflammatory pathways are up-regulated in OBCs. To separately examine molecular signatures from tumor, stroma and normal compartments, we used non-negative matrix factorization (NMF) analyses to virtually dissect bulk tumor expression data and identified 14 factors including 3 factors associated with normal tissues, 1 factor associated with stroma and 1 factor associated with tumor infiltrating leukocytes (TILs). By examining the correlation between pathway gene expression and NMF factors, we inferred that DE pathways such as fatty acid metabolism, bile acid biosynthesis, and epithelial-to-mesenchymal transition (EMT) were mainly active in stromal and normal tissue compartments. The TIL factor was significantly enriched in Asian BCs relative to Caucasian BCs with the highest TIL factor weight observed in Asian OBCs. Using gene expression signatures representing distinct types of TILs, we classified the combined cohort into three subtypes of varying TIL activities. Consistent with results from the NMF analysis, the TIL-high subtype is also significantly enriched in Asian BCs relative to Caucasian BCs. To our knowledge, this is the first large-scale multi-omics study of Asian breast cancer. Comparative analyses of multi-omics profiles from Asian and primarily Caucasian BC cohorts identified distinguishing molecular signatures associated with Asian BCs. Further, many signatures appeared to be specific to non-tumor compartments within bulk tumor, indicating that young Asian BCs may harbor distinctive tumor microenvironment. Citation Format: Yeon Hee Park, Ying Ding, Soo-Hyeon Lee, Hae Hyun Jung, Woosung Chung, Soonweng Cho, Jin-Ho Kim, Shibing Deng, Yoon-la Choi, Julio Fernandez, Se Kyung Lee, Seok Won Kim, Jeong Eon Lee, Ji-Yeon Kim, Jin Seok Ahn, Young-Hyuck Im, Seok Jin Nam, Woong-Yang Park, Zhengyan Kan. Comparative analyses of multi-omics profiles reveal distinctive molecular signatures of young Asian breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3370. doi:10.1158/1538-7445.AM2017-3370

Abstract 4231: Combination of epigenetic, differentiation and DNA damaging agents induce tumor cell death and stem cell depletion in breast cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The histone deacetylase inhibitor, entinostat, is a new-generation epigenetic drug, which has recently demonstrated notable clinical efficacy when used in combination with standard therapy. Retinoids induce differentiation in various types of stem cells. However, its delivery to patients is challenging because of its rapid metabolism. Also, epigenetic changes in the retinoic acid receptors often render cancer cells retinoid-resistant. We have shown that a combination of entinostat, all-trans retinoic acid (ATRA) and doxorubicin causes significant regression of xenografts of triple negative breast cancer (TNBC) cells and investigated the mechanism underlying the effectiveness of this combination therapy. Combinations of entinostat, retinoic acid and doxorubicin were optimal in causing tumor regression in triple negative breast cancer xenografts. Gene expression analysis of treated TNBC cells identified genes most effectively reprogrammed by entinostat and doxorubicin (ED) combination therapy. These genes are involved in cell cycle arrest, inflammation and differentiation, which are related to better survival outcome in patients. Entinostat sensitizes the cells to doxorubicin-induced growth arrest, resulting in increased apoptosis and necrosis. Adding ATRA to ED regulated interferon genes and members of the cancer/testis antigens (CTA) and tripartite motif (TRIM) family of proteins and induces inflammation in nude mice. Entinostat/ATRA/dox therapy was most effective to target breast cancer stem cells (BCSC) in limiting dilution assays of growth in mammary fat pads. The epithelium-specific ETS transcription factor-1 (ESE-1 or ELF3), known to regulate cellular proliferation and differentiation mediates the epigenetic differentiation effect. Patient-derived distant metastases responded to entinostat/ATRA/dox therapy in culture. Thus, the combination therapy may have significant effects in decreasing both local and metastatic growth in breast cancer, especially in decreasing recurrence by targeting breast cancer stem cells. Citation Format: Vanessa F. Merino, Nguyen Nguyen, Helen Sadik, Soonweng Cho, Leslie Cope, Xian C. Zhou, Zhe Zhang, Qian Chen, Duojia Pan, David L. Huso, Syed Ali, Christina Adams, Balazs Győrffy, Saraswati Sukumar. Combination of epigenetic, differentiation and DNA damaging agents induce tumor cell death and stem cell depletion in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4231. doi:10.1158/1538-7445.AM2015-4231