TCGA Breast Cancer Research Articles

Estrogen-regulated PTTG1 promotes breast cancer progression by regulating cyclin kinase expression

BackgroundThe present study aims to investigate the effects of pituitary tumor transforming gene (PTTG) 1 on breast cancer and its underlying mechanism.MethodsGEO data set was applied to analyze the relationship between PTTG1 and survival status and the TCGA breast cancer dataset was used to explore its possible targets. The stable cell lines including PTTG1 knockdown cells, estrogen receptor (ESR) 1 knockdown cells, and PTTG1 overexpression cells were constructed. MTT was used to determine cell viabilities. Propidium iodide (PI) staining and flow cytometry were used to analyze the cell cycle. Quantitative polymerase chain reaction (qPCR) was employed to determine the mRNA expressions. Points mutations and luciferase reporter assays were used to determine the binding sites of estrogen.ResultsPTTG1 was associated with poor survival rates in breast cancer. In vitro study demonstrated that PTTG1 affected cell viabilities of MCF7 and T47D cells. Besides, PTTG1 affected cell cycle arrest of breast cancer cells. Overexpression of PTTG1 led to more breast cancer cells distributed in S phase. The levels of PTTG1 were associated with estrogen and further results showed that the levels of PTTG1 were positively correlated to tamoxifen resistance. Two genes including CCNA2 and CCNB2 were identified to be possible targets of PTTG1.ConclusionEstrogen-regulated PTTG1 promotes the development of breast cancer cells by the regulation of the cell cycle.

Isoform-specific promotion of breast cancer tumorigenicity by TBX3 involves induction of angiogenesis

TBX3 is a member of the highly conserved family of T-box transcription factors involved in embryogenesis, organogenesis and tumor progression. While the functional role of TBX3 in tumorigenesis has been widely studied, less is known about the specific functions of the different isoforms (TBX3iso1 and TBX3iso2) which differ in their DNA-binding domain. We therefore sought to investigate the functional consequence of this highly conserved splice event as it relates to TBX3-induced tumorigenesis. By utilizing a nude mouse xenograft model, we have identified differential tumorigenic potential between TBX3 isoforms, with TBX3iso1 overexpression more commonly associated with invasive carcinoma and high tumor vascularity. Transcriptional analysis of signaling pathways altered by TBX3iso1 and TBX3iso2 overexpression revealed significant differences in angiogenesis-related genes. Importantly, osteopontin (OPN), a cancer-associated secreted phosphoprotein, was significantly up-regulated with TBX3iso1 (but not TBX3iso2) overexpression. This pattern was observed across three non/weakly-tumorigenic breast cancer cell lines (21PT, 21NT, and MCF7). Up-regulation of OPN in TBX3iso1 overexpressing cells was associated with induction of hyaluronan synthase 2 (HAS2) expression and increased retention of hyaluronan in pericellular matrices. These transcriptional changes were accompanied by the ability to induce endothelial cell vascular channel formation by conditioned media in vitro, which could be inhibited through addition of an OPN neutralizing antibody. Within the TCGA breast cancer cohort, we identified an 8.1-fold higher TBX3iso1 to TBX3iso2 transcript ratio in tumors relative to control, and this ratio was positively associated with high-tumor grade and an aggressive molecular subtype. Collectively, the described changes involving TBX3iso1-dependent promotion of angiogenesis may thus serve as an adaptive mechanism within breast cancer cells, potentially explaining differences in tumor formation rates between TBX3 isoforms in vivo. This study is the first of its kind to report significant functional differences between the two TBX3 isoforms, both in vitro and in vivo.

Abstract P1-06-02: Characterization of gene- and sample-level APOBEC mutagenesis enrichment with respect to intrinsic subtypes, tumor mutational burden, and immune composition in breast cancer

Abstract Introduction: APOlipoprotein B mRNA Editing enzyme Catalytic peptide-like, APOBEC, is a family of innate immune enzymes that catalyze cytosine to uracil deamination in single-stranded DNA, which lead to mutations in preferred trinucleotide contexts. APOBEC mutagenesis portends a worse prognosis in several breast cancer subtypes and associates with increased tumor mutational burden (TMB). Specific mutations enriched with APOBEC mutation signatures and immune phenotypes are not well described. The purpose of our study was to identify genes enriched for APOBEC mutagenesis and determine their relationship to intrinsic subtypes, TMB, and the immune composition of the transcriptome in breast cancers. Methods: We queried TCGA breast cancer database involving 980 breast cancer samples. We used consensus mutation calls from four somatic mutation callers (MuTect2, VarScan2, MuSE, and SomaticSniper). APOBEC mutagenesis scores were calculated for each tumor using a published method (Nik-Zainal et al. 2012) and a modified version thereof. The score was calculated at the gene-level, to identify genes enriched for APOBEC mutagenesis, and the cancer sample-level to investigate the relationship of APOBEC mutagenesis scores with intrinsic subtypes (using PAM50), APOBEC3A and 3B expression levels, and TMB. We also undertook differential expression analysis of CIBERSORT gene signatures and individual genes from the innate immune system, IFN, TGFB, WNT, and immune checkpoints with respect to common somatic mutations in APOBEC enriched genes. Results: The genes with the strongest statistical evidence for enrichment of APOBEC mutagenesis were PIK3CA, TP53, MUC16, and TTN. Among three common somatic mutations in PIK3CA (E542K, E545K, H1047R), the APOBEC score was only observed to be associated with E542K (q=2.47 × 10−2) and E545K (q=9.81 × 10−6) mutations. APOBEC mutagenesis revealed a high degree of enrichment across all breast cancer intrinsic subtypes, 75% in luminal A, 68% in luminal B, 91% in HER2 enriched, 73% in basal, and 89% in normal-like phenotypes. APOBEC mutagenesis scores were observed to significantly associate with APOBEC3A expression (q=1.1 × 10−9) and as a trend of association with APOBEC3B expression (q=1.62 × 10−1). A strong association was observed between APOBEC mutagenesis score and elevated TMB (p<2 × 10−16). No checkpoint genes (i.e. CTLA-4, PD-1, and PD-L1) were observed to be significantly overexpressed among those tumors with E542K and E545K PIK3CA mutations. The CIBERSORT mast cell gene signature (q=1.32 × 10−2) and the individual genes TGFB3 (q=5 × 10−3), ROR (q=7.91 × 10−3), and WNT5A (q=1.92 × 10-2) were overexpressed in breast tumors with E542K PIK3CA mutations. Conclusion: APOBEC mutagenesis patterns are seemingly prevalent across all breast cancer intrinsic subtypes and associate with elevated TMB and mutations in the PIK3CA helical loop domain and TP53 gene in TCGA breast cancer samples. Cancers with PIK3CA mutations in the helical loop domain did not demonstrate checkpoint gene overexpression, but were associated with other immunosuppressive features (e.g. TGFB3 and WNT5A overexpression). Investigation into innate immune cell signaling, mast cell immune signaling, and immunosuppressive expression profiles in APOBEC mutated breast cancers with and without PIK3CA mutations may help to identify novel immune targets to combine with PI3K inhibitors in breast cancer. Citation Format: Jeremy Force, Xiaodi Qin, Dadong Zhang, P. Kelly Marcom, Jeffrey Marks, Mary Love Taylor, Carey Anders, Kouros Owzar, Jichun Xie. Characterization of gene- and sample-level APOBEC mutagenesis enrichment with respect to intrinsic subtypes, tumor mutational burden, and immune composition in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-06-02.

Abstract P4-09-01: Clinical sequencing characterizes the genomic landscape and actionable mutations of Chinese breast cancer

Abstract Here, we prospectively collected what is currently the largest Chinese breast cancer cohort of 1,143 patients for clinical sequencing and performed integrated analysis of their clinical and genomic characteristics. All samples were collected via needle biopsy. A custom-designed genetic panel was used in this study. The panel was a hybridization capture-based assay, including 484 genes that are targets of approved and experimental therapies as well as frequently mutated genes in breast cancer. The panel was designed for detecting mutations and small insertions and deletions. The highest prevalence of breast cancer-related variations observed in our Chinese cohort was TP53 mutations (53%), followed by PIK3CA (19%), NF1 (10%), GATA3 (9%) and KMT2C (9%) mutations. The hotspot mutations (with frequencies higher than 2%) in Chinese breast cancer included PIK3CA p. H1047R (10%), AKT1 p. E17K (4%), KMT2C p. K2797fs (2%) and TP53 p. R248Q (2%). PIK3CA and AKT1 mutations were found to be especially enriched in the Luminal (HER2-) subtype, NF1 and ERBB2 mutations were enriched in Luminal (HER2+) subtype, and PTEN mutations were enriched in triple negative breast cancer. In addition, there was significant disparity of mutation load among the different subtypes, suggesting a high mutation load in triple negative breast cancer. We further investigated the differences in mutational features between the Chinese cohort and foreign published cohorts, especially the TCGA and MSKCC breast cancer datasets, and revealed the distinction mainly existed in breast cancer of the HR+/HER2- subtype, while the other subtypes showed a similar mutation prevalence. We evaluated 9 canonical signaling pathways with frequent oncogenic alterations. The pathways that we analyzed included the cell cycle signaling, Hippo signaling, Notch signaling, PI-3-Kinase (PI3K) signaling, β-catenin/Wnt signaling, receptor-tyrosine (RTK)/RAS/MAP-Kinase (RTK-RAS) signaling, p53 signaling, TGF-β signaling and Myc signaling pathways. Alterations in the RTK-RAS pathway were exclusive to those in the PI3K pathway in hormone receptor (HR)-positive breast cancer and co-occurred with those in the Notch pathway in human epidermal growth factor receptor-2 (HER2)-positive, HR- and HER2-positive breast cancer. PI3K signaling pathway mutations were identified as a driving factor, and our finding revealed the competitively oncogenic role of mutations in the RTK-RAS pathway, especially in the HR+/HER2- subtype. Furthermore, we identified mutations in the P53 (53% versus 31%, p < 0.001), RTK-RAS (31% versus 18%, p < 0.001), Notch (17% versus 11%, p < 0.001), WNT (7% versus 1%, p < 0.001) and Hippo (2% versus 0%, p < 0.001) pathways that were more prevalent in our Chinese cohort than in the Caucasian cohort. We further explored the potential actionable targets in Chinese patients with breast cancer who might benefit from our sequencing. The OncoKB classification system was used to stratify levels of genomic biomarkers in the different subtypes. Over 32% of breast cancer patients could be genomically matched to at least one actionable biomarker. Notably, NF1 and NOTCH1 mutations were markedly enriched in Chinese patients, suggesting the potential for novel treatment strategies for breast cancer. Taken together, our study comprehensively revealed the characteristics of mutations in Chinese breast cancer, improving our understanding of the mutational diversity among different molecular subtypes, identifying potential treatment biomarkers and leading to systematic genomic studies and novel clinical trials. Citation Format: Guan-Tian Lang, Jin-Xiu Shi, Yi-Zhou Jiang, Xiao-Guang Li, Yu-Chen Pei, Fang-Lin Zhang, Chen-Hui Zhang, Ding Ma, Yi Xiao, Ke-Da Yu, Peng-Chen Hu, Ming-Liang Jin, Hai Wang, Yun-Song Yang, Xuan Luo, Qi Hong, Feng Qiao, Wei-Li Sun, Meng-Zhu Xue, Shi-Ping Li, A-Yong Cao, Zhong-Hua Wang, Jia-Xin Zhang, Gen-Hong Di, Peng Wang, Da-Qiang Li, Xin Hu, Wei Huang, Zhi-Ming Shao. Clinical sequencing characterizes the genomic landscape and actionable mutations of Chinese breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-01.

Abstract P3-11-21: Identification and characterization of novel splice variants in triple-negative breast cancer

Abstract Splicing deregulation is a major hallmark of cancer, affecting progression, metastasis and therapy resistance. Multiple studies have demonstrated the oncogenic activity of specific splicing events and splicing factors, including SRSF1, SRSF2, ESRP1, and RBFOX1, in human and animal models. As such, cancer-associated splicing deregulation may be a novel source of clinically-applicable biomarkers and therapeutic targets, particularly for treatment-insensitive cancers such as TNBC. Here, we identified and experimentally characterized novel splicing changes in triple negative breast cancer by analyzing TCGA breast cancer RNA-seq data using our software platform SpliceCore. SpliceCore integrates machine learning (ML) algorithms with high-performance computing to analyze large RNA-Seq datasets to predict biologically relevant splicing events. Our analysis of TCGA BRCA datasets has revealed several previously unknown splicing events specific to TNBC patient cohorts, that is subsequently experimentally validated in TNBC cell lines. We provide biological evidence for such TNBC specific splice-switch both at the RNA and protein level. We provide evidence for SpliceCore predicted splice-switching oligonucleotides (SSO) and verify their biological activity in TNBC cell lines. Further, functional studies using potent and specific SSOs against the top candidates reveal a critical role for some of these novel isoforms in TNBC. Together, we provide a proof of concept study using SpliceCore to identify, design, and target novel splicing changes for subsequent clinical translation and therapeutic development. Citation Format: Gayatri Arun, Priyanka Dhingra, Robin Munch, Vanessa Fredrick, Martin Akerman. Identification and characterization of novel splice variants in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-21.

Abstract P1-03-08: In vivo investigation of the Duffy antigen receptor for chemokines (DARC/ACKR1) and tumor-infiltrating immune cells in the breast tumor microenvironment

Abstract Recruitment of immune cells and subsequent infiltration of these cell populations into the tumor microenvironment (TME) can influence disease prognosis, and potential treatments for patients. Through studying the TME of breast tumors, we have investigated factors that modify immune cell recruitment and infiltration into the tumor area. In our recently published work, we analyzed the TCGA breast cancer (BC) cohort, where we have identified the Duffy Antigen Receptor for Chemokines / Atypical Chemokine Receptor 1 (DARC/ACKR1) as a putative driver of immune cell recruitment and infiltration to the breast TME. DARC/ACKR1 is an atypical chemokine receptor that promiscuously binds both the CC and CXC classes of chemokines. On the surface of red blood cells in circulation, DARC/ACKR1 maintains homeostatic levels of chemokines in circulation by binding chemokines when levels are high, and releasing them back into circulation when levels lower. In tissues, DARC/ACKR1 participates in chemokine transcytosis, working to move chemokines across the cell layer to establish chemokine gradients necessary for recruitment of respective immune cell populations. Through in silico analysis of TCGA BC cohort data, we performed CIBERSORT TME deconvolution methods to approximated levels of 22 tumor-associated immune cell populations. We showed that as DARC/ACKR1 expression increased in breast tumors, we also saw a significant increase in number of tumor-associated immune cells (p<0.0001). When looking at the specific cell populations, we found that B cells, T cells, monocytes and macrophage populations were significantly different between high, mid and low DARC/ACKR1 expressing tumors. To continue investigation of these findings in vivo, we developed a novel DARC/ACKR1 breast cancer transgenic mouse model. Briefly DARC/ACKR1 -/- mice were crossed with the C3(1)Tag breast cancer transgenic mouse model. Breast cancer Tg+; DARC/ACKR1 +/- males were then backcrossed to DARC/ACKR1 -/- females, to generate target female mice that were BC Tg+, and either DARC/ACKR1 +/- or DARC/ACKR1 -/-. Disease progression in the C3(1)Tag line follows a similar pattern to ductal carcinoma in situ (DCIS), through progression to advanced invasive disease. All mice develop DCIS beginning at approximately 12 weeks of age, and 100% of mice are affected with invasive disease by 6 months of age. We have collected tissue from mice from DCIS stage of disease through invasive disease. Blood was collected from each mouse, and the plasma was used to quantify the circulating chemokine profile. Tumors and mammary tissue were collected from each mouse and weighed, alongside spleen, liver, and potential metastatic tumor sites. Tumors and mammary glands were processed for immunohistochemistry and immunofluorescent staining procedures, where DARC/ACKR1 expression, immune cells, structural markers and chemokines of interest were assayed. We also took representative sections from each genotype of interest for Hyperion imaging mass cytometry analysis, where we can simultaneously analysis 27 different immune, structural and other markers of interest. From our preliminary results from in silico TCGA analysis, we expect to see changes in immune cell recruitment and infiltration, specifically in quantity of immune cells recruited and infiltrating, and in the composition of the tumor associated immune cells between our DARC/ACKR1 expression or non-expressing BC Tg+ mice. Citation Format: Rachel Martini, Lisa Newman, Nancy Manley, Melissa Davis. In vivo investigation of the Duffy antigen receptor for chemokines (DARC/ACKR1) and tumor-infiltrating immune cells in the breast tumor microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-08.

Changes in DNA Damage Repair Gene Expression and Cell Cycle Gene Expression Do Not Explain Radioresistance in Tamoxifen-Resistant Breast Cancer.

Tamoxifen-induced radioresistance, reported in vitro, might pose a problem for patients who receive neoadjuvant tamoxifen treatment and subsequently receive radiotherapy after surgery. Previous studies suggested that DNA damage repair or cell cycle genes are involved, and could therefore be targeted to preclude the occurrence of cross-resistance. We aimed to characterize the observed cross-resistance by investigating gene expression of DNA damage repair genes and cell cycle genes in estrogen receptor-positive MCF-7 breast cancer cells that were cultured to tamoxifen resistance. RNA sequencing was performed, and expression of genes characteristic for several DNA damage repair pathways was investigated, as well as expression of genes involved in different phases of the cell cycle. The association of differentially expressed genes with outcome after radiotherapy was assessed in silico in a large breast cancer cohort. None of the DNA damage repair pathways showed differential gene expression in tamoxifen-resistant cells compared to wild-type cells. Two DNA damage repair genes were more than two times upregulated (NEIL1 and EME2), and three DNA damage repair genes were more than two times downregulated (PCNA, BRIP1, and BARD1). However, these were not associated with outcome after radiotherapy in the TCGA breast cancer cohort. Genes involved in G1, G1/S, G2, and G2/M phases were lower expressed in tamoxifen-resistant cells compared to wild-type cells. Individual genes that were more than two times upregulated (MAPK13) or downregulated (E2F2, CKS2, GINS2, PCNA, MCM5, and EIF5A2) were not associated with response to radiotherapy in the patient cohort investigated. We assessed the expression of DNA damage repair genes and cell cycle genes in tamoxifen-resistant breast cancer cells. Though several genes in both pathways were differentially expressed, these could not explain the cross-resistance for irradiation in these cells, since no association to response to radiotherapy in the TCGA breast cancer cohort was found.

Differential Allele-Specific Expression Uncovers Breast Cancer Genes Dysregulated by Cis Noncoding Mutations.

Identifying cancer-relevant mutations in noncoding regions is challenging due to the large numbers of such mutations, their low levels of recurrence, and difficulties in interpreting their functional impact. To uncover genes that are dysregulated due to somatic mutations in cis, we build upon the concept of differential allele-specific expression (ASE) and introduce methods to identify genes within an individual's cancer whose ASE differs from what is found in matched normal tissue. When applied to breast cancer tumor samples, our methods detect the known allele-specific effects of copy number variation and nonsense-mediated decay. Further, genes that are found to recurrently exhibit differential ASE across samples are cancer relevant. Genes with cis mutations are enriched for differential ASE, and we find 147 potentially functional noncoding mutations cis to genes that exhibit significant differential ASE. We conclude that differential ASE is a promising means for discovering gene dysregulation due to cis noncoding mutations.

FGFR3 signaling and function in triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) accounts for 16% of breast cancers and represents an aggressive subtype that lacks targeted therapeutic options. In this study, mass spectrometry (MS)-based tyrosine phosphorylation profiling identified aberrant FGFR3 activation in a subset of TNBC cell lines. This kinase was therefore evaluated as a potential therapeutic target.MethodsMS-based tyrosine phosphorylation profiling was undertaken across a panel of 24 TNBC cell lines. Immunoprecipitation and Western blot were used to further characterize FGFR3 phosphorylation. Indirect immunofluorescence and confocal microscopy were used to determine FGFR3 localization. The selective FGFR1–3 inhibitor, PD173074 and siRNA knockdowns were used to characterize the functional role of FGFR3 in vitro. The TCGA and Metabric breast cancer datasets were interrogated to identify FGFR3 alterations and how they relate to breast cancer subtype and overall patient survival.ResultsHigh FGFR3 expression and phosphorylation were detected in SUM185PE cells, which harbor a FGFR3-TACC3 gene fusion. Low FGFR3 phosphorylation was detected in CAL51, MFM-223 and MDA-MB-231 cells. In SUM185PE cells, the FGFR3-TACC3 fusion protein contributed the majority of phosphorylated FGFR3, and largely localized to the cytoplasm and plasma membrane, with staining at the mitotic spindle in a small subset of cells. Knockdown of the FGFR3-TACC3 fusion and wildtype FGFR3 in SUM185PE cells decreased FRS2, AKT and ERK phosphorylation, and induced cell death. Knockdown of wildtype FGFR3 resulted in only a trend for decreased proliferation. PD173074 significantly decreased FRS2, AKT and ERK activation, and reduced SUM185PE cell proliferation. Cyclin A and pRb were also decreased in the presence of PD173074, while cleaved PARP was increased, indicating cell cycle arrest in G1 phase and apoptosis. Knockdown of FGFR3 in CAL51, MFM-223 and MDA-MB-231 cells had no significant effect on cell proliferation. Interrogation of public datasets revealed that increased FGFR3 expression in breast cancer was significantly associated with reduced overall survival, and that potentially oncogenic FGFR3 alterations (eg mutation and amplification) occur in the TNBC/basal, luminal A and luminal B subtypes, but are rare.ConclusionsThese results indicate that targeting FGFR3 may represent a therapeutic option for TNBC, but only for patients with oncogenic FGFR3 alterations, such as the FGFR3-TACC3 fusion.AQe7iP6srQZx6BoH-5jRjTVideo abstract.

Definition of a New Metric With Mutual Exclusivity and Coverage for Identifying Cancer Driver Modules

Identification of cancer driver modules or pathways is a key step in understanding cancer pathogenesis and exploring patient-specific treatments. Numerous studies have shown that some genes with low mutation frequency are also important for the cancer progression, while previous research have focused on identifying high-frequency mutation genes. In this study, we propose a new framework with a new metric to identify driver modules with low-frequency mutation genes, called iCDModule. Inspired by the gravity model, we integrate the coverage and mutual exclusivity in mutation information, define a new metric between gene pairs, called mutation impact distance, to help identifying potential driver genes sets, including those have extremely low mutation rates but play an important role in functional networks. A genetic network is constructed by combining the defined mutation impact distance and then the driver module identification problem is formalized as the maximum clique solution problem, and an improved ant colony optimization algorithm is used to solve it. iCDModule is applied to TCGA breast cancer, glioblastoma, ovarian cancer to test performance. Experiments show that it can accurately identify known cancer driver modules and pathways, and also detect driver modules containing low-frequency mutation genes. iCDModule is significantly better than other existing methods in identifying driver modules.

Identification of key microRNAs involved in tumorigenesis and prognostic microRNAs in breast cancer.

Breast cancer is a commonly diagnosed cancer in women, and one of the leading causes of cancer-related death among female patients However, the key microRNAs involved in its tumorigenesis and microRNAs of prognostic values have not been fully understood. In the present study, we aimed to perform a systematic analysis of microRNA expression profiles to identify some key microRNAs associated with tumor initiation and prognosis. Using TCGA breast cancer datasets, we identified 110 differentially expressed microRNAs. The functional enrichment analysis of the upregulated microRNAs revealed signaling transduction pathways, such as Notch and Wnt signaling pathway, and metabolism-related pathways such as sugar and nucleotide sugar metabolism, and oxidative stress response. Moreover, multivariable Cox model based on three variables of hsa-mir-130a, hsa-mir-3677, and hsa-mir-1247 stratified patients into high-risk and low-risk groups, which showed significant prognostic difference. In addition, we also tested the performance of this model in patient cohorts of any specific breast cancer subtypes or different TNM stages. The high performance in risk prediction was also observed in all of breast cancer subtypes and TNM stages. We also observed that there were highly possible interactions between hsa-mir-130a and seven target genes. Among these target genes, VAV3 and ESR1 were predicted as the target genes of hsa-mir-130a, suggesting that hsa-mir-130a may function by regulating the expression of VAV3 and ESR1 in breast cancer. In conclusion, the stratification based on the multivariable Cox model showed high performance in risk prediction. The dysregulated microRNAs and prognostic microRNAs greatly improved our understanding of the microRNA-related molecular mechanism underlying breast cancer.

Protein Phosphatase 1H, Cyclin-Dependent Kinase Inhibitor p27, and Cyclin-Dependent Kinase 2 in Paclitaxel Resistance for Triple Negative Breast Cancers.

PurposePaclitaxel is a cytotoxic chemotherapy commonly used in patients with triple negative breast cancer (TNBC); however, the resistance to paclitaxel is a cause of poor response in the patients. The aim of this study was to examine the role of protein phosphatase 1H (PPM1H) in paclitaxel resistance in breast cancer patients.MethodsTo investigate the function of PPM1H in paclitaxel treatment, we conducted in vitro assays and molecular experiments using a stable cell line (MDA-MB-231) in which PPM1H is overexpressed. We also performed molecular analyses on patient tissue samples. Molecular expression related to PPM1H in breast cancer patients was analyzed using TCGA data.ResultsWe investigated whether PPM1H was associated with paclitaxel resistance in breast cancer. PPM1H expression was upregulated in breast cancer cells treated with paclitaxel. We also observed that overexpression of PPM1H in breast cancer cells resulted in increased sensitivity to paclitaxel in vitro. Additionally, paclitaxel treatment induced dephosphorylation of cyclin-dependent kinase (CDK) inhibitor p27 (p27), which was more evident in PPM1H-overexpressing cells. To understand how upregulation of PPM1H increases paclitaxel sensitivity, we determined the levels of p27, phospho-p27, and CDK2, since CDK2 exerts antagonistic effects against PPM1H on p27 phosphorylation. The patient-derived xenograft (PDX) tumors that did not respond to paclitaxel showed increased levels of CDK2 and phospho-p27 and decreased levels of total p27 compared to the other breast tumor tissues. The use of dinaciclib, a selective CDK inhibitor, significantly inhibited tumor growth in the PDX model.ConclusionCDK2 kinase activity was significantly upregulated in basal breast cancer tumors and was negatively correlated with p27 protein levels in the TCGA breast cancer dataset, suggesting that targeting CDK2 may be an effective treatment strategy for TNBC patients.

Loss of SIRT4 promotes the self-renewal of Breast Cancer Stem Cells.

Rationale: It has been proposed that cancer stem/progenitor cells (or tumor-initiating cells, TICs) account for breast cancer initiation and progression. Sirtuins are nicotinamide adenine dinucleotide (NAD+)-dependent class-III histone deacetylases and mediate various basic biological processes, including metabolic homeostasis. However, interplay and cross-regulation among the sirtuin family are not fully understood. As one of the least studied sirtuin family members, the mitochondrial sirtuin SIRT4 is a tumor suppressor gene in various cancers. However, its role in cancer stemness, as well as initiation and progression of breast cancer, remains unknown.Methods: The expression of SIRT4 in breast cancer was analyzed using the TCGA breast cancer database and 3 GSEA data. Normal breast epithelial cells MCF10A and breast cancer cell lines MCF-7, MDA-MB-231, BT549, MDA-MB-468 were used to establish SIRT4 gene knockdown and corresponding overexpression cells. Identified MTT cytotoxicity assays, cell invasion and motility assay, sorting of SP, confocal immunofluorescence microscopy, mouse mammary stem cell analysis, glutamine and glucose production, clonogenic and sphere-formation assay, mass spectrometric metabolomics analysis and ChIP-seq to further explore SIRT4 biological role in breast cancer.Results: We elucidated a novel role for SIRT4 in the negative regulation of mammary gland development and stemness, which is related to the mammary tumorigenesis. We also uncovered an inverse correlation between SIRT4 and SIRT1. Most importantly, SIRT4 negatively regulates SIRT1 expression via repressing glutamine metabolism. Besides, we identified H4K16ac and BRCA1 as new prime targets of SIRT4 in breast cancer.Conclusions: These results demonstrate that SIRT4 exerts its tumor-suppressive activity via modulating SIRT1 expression in breast cancer and provide a novel cross-talk between mitochondrial and nuclear sirtuins.

1917P - Luminal B breast cancer prognosis prediction by comprehensive analysis of Homeobox genes

BackgroundHomeobox (HOX) family consists of 39 genes which act as master regulators in embryonic development. Each of the genes is also known to play key roles in progression of breast cancer, including epithelial to mesenchymal transition, tumor angiogenesis and endocrine therapy resistance. Although there are numerous reports on individual HOX genes and cancer, none of them have comprehensively analyzed the whole gene family. Since HOX genes strongly coordinate within the family during the embryonic period, we considered that the analysis of the whole HOX family is also indispensable in breast cancer. MethodsWe collected 702 breast cancer data from four publicly available array datasets (GSE11121, GSE7390, GSE3494, GSE2990) and performed unsupervised hierarchal clustering into two clusters by the expression of HOX genes. We constructed model formulas for cluster prediction by dividing the samples into learning and validation groups. We used three machine learning methods: support-vector machine (SVM), neural network and Bayes. The model formulas were validated by validation samples. We also used 512 TCGA breast cancer data to calculate covariations of the genes in breast cancer. ResultsBy the clustering of four array datasets, the DFS of the two clusters in PAM50-classified luminal B patients were statistically different (p=0.016), and the gene ontology analysis revealed that the Wnt pathway was activated in the poor prognostic cluster. All cluster prediction models for luminal B sample achieved accuracies of over 90%. From TCGA breast cancer data, we found that HOX genes covariate the most with other HOX genes, especially within the chromosomally proximal groups. ConclusionsComprehensive analysis of the whole HOX family lead to the prediction of luminal B breast cancer prognosis. Considering that Wnt signaling controls HOX genes during the embryonic stage, we suppose a Wnt pathway activated, poor prognostic subgroup in luminal B breast cancer which can be identified by the expression of HOX genes. The cluster prediction model by machine learning was acceptable for its future adaptation in clinical settings. We also proved that HOX genes strongly covariate within the gene family in cancer, not only during the embryonic stage. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Genetic determinants of cellular addiction to DNA polymerase theta

Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy.

Integrated Transcriptome and Pathway Analyses Revealed Multiple Activated Pathways in Breast Cancer.

Breast cancer (BC) is the leading cause of cancer-related death in women. Therefore, a better understanding of BC biology and signaling pathways might lead to the development of novel biomarkers and targeted therapies. Although a number of transcriptomic studies have been performed on breast cancer patients from various geographic regions, there are almost no such comprehensive studies performed on breast cancer from patients in the gulf region. This study aimed to provide a better understanding of the altered molecular networks in BC from the gulf region. Herein, we compared the transcriptome of BC to adjacent normal tissue from six BC patients and identified 1,108 upregulated and 518 downregulated transcripts. A selected number of genes from the RNA-Seq analysis were subsequently validated using qRT-PCR. Differentially expressed (2.0-fold change, adj. p < 0.05) transcripts were subjected to ingenuity pathway analysis, which revealed a myriad of affected signaling pathways and functional categories. Activation of ERBB2, FOXM1, ESR1, and IGFBP2 mechanistic networks was most prominent in BC tissue. Additionally, BC tissue exhibited marked enrichment in genes promoting cellular proliferation, migration, survival, and DNA replication and repair. The presence of genes indicative of immune cell infiltration and activation was also observed in BC tissue. We observed high concordance [43.5% (upregulated) and 62.1% (downregulated)] between differentially expressed genes in our study group and those reported for the TCGA BC cohort. Our data provide novel insight on BC biology and suggest common altered molecular networks in BC in this geographic region. Our data suggest future development of therapeutic interventions targeting those common signaling pathways.

A functional role for the cancer disparity-linked genes, CRY\u03b2B2 and CRY\u03b2B2P1, in the promotion of breast cancer

BackgroundIn the USA, the breast cancer mortality rate is 41% higher for African-American women than non-Hispanic White women. While numerous gene expression studies have classified biological features that vary by race and may contribute to poorer outcomes, few studies have experimentally tested these associations. CRYβB2 gene expression has drawn particular interest because of its association with overall survival and African-American ethnicity in multiple cancers. Several reports indicate that overexpression of the CRYβB2 pseudogene, CRYβB2P1, and not CRYβB2 is linked with race and poor outcome. It remains unclear whether either or both genes are linked to breast cancer outcomes. This study investigates CRYβB2 and CRYβB2P1 expression in human breast cancers and breast cancer cell line models, with the goal of elucidating the mechanistic contribution of CRYβB2 and CRYβB2P1 to racial disparities.MethodsCustom scripts for CRYβB2 or CRYβB2P1 were generated and used to identify reads that uniquely aligned to either gene. Gene expression according to race and tumor subtype were assessed using all available TCGA breast cancer RNA sequencing alignment samples (n = 1221). In addition, triple-negative breast cancer models engineered to have each gene overexpressed or knocked out were developed and evaluated by in vitro, biochemical, and in vivo assays to identify biological functions.ResultsWe provide evidence that CRYβB2P1 is expressed at higher levels in breast tumors compared to CRYβB2, but only CRYβB2P1 is significantly increased in African-American tumors relative to White American tumors. We show that independent of CRYβB2, CRYβB2P1 enhances tumorigenesis in vivo via promoting cell proliferation. Our data also reveal that CRYβB2P1 may function as a non-coding RNA to regulate CRYβB2 expression. A key observation is that the combined overexpression of both genes was found to suppress cell growth. CRYβB2 overexpression in triple-negative breast cancers increases invasive cellular behaviors, tumor growth, IL6 production, immune cell chemoattraction, and the expression of metastasis-associated genes. These data underscore that both CRYβB2 and CRYβB2P1 promote tumor growth, but their mechanisms for tumor promotion are likely distinct.ConclusionsOur findings provide novel data emphasizing the need to distinguish and study the biological effects of both CRYβB2 and CRYβB2P1 as both genes independently promote tumor progression. Our data demonstrate novel molecular mechanisms of two understudied, disparity-linked molecules.

Migration rather than proliferation transcriptomic signatures are strongly associated with breast cancer patient survival

The efficacy of prospective cancer treatments is routinely estimated by in vitro cell-line proliferation screens. However, it is unclear whether tumor aggressiveness and patient survival are influenced more by the proliferative or the migratory properties of cancer cells. To address this question, we experimentally measured proliferation and migration phenotypes across more than 40 breast cancer cell-lines. Based on the latter, we built and validated individual predictors of breast cancer proliferation and migration levels from the cells’ transcriptomics. We then apply these predictors to estimate the proliferation and migration levels of more than 1000 TCGA breast cancer tumors. Reassuringly, both estimates increase with tumor’s aggressiveness, as qualified by its stage, grade, and subtype. However, predicted tumor migration levels are significantly more strongly associated with patient survival than the proliferation levels. We confirmed these findings by conducting siRNA knock-down experiments on the highly migratory MDA-MB-231 cell lines and deriving gene knock-down based proliferation and migration signatures. We show that cytoskeletal drugs might be more beneficial in patients with high predicted migration levels. Taken together, these results testify to the importance of migration levels in determining patient survival.

Abstract 2926: Nomination and characterization of TTK for radiosensitization in basal-like breast cancers

Abstract Background: Increased rates of locoregional recurrence have been observed in basal-like breast cancer despite the use of radiation therapy (RT), therefore approaches that result in radiosensitization of basal-like breast cancer are critically needed. Studies detailing the poor response of basal-like tumors to adjuvant RT underscore the biologic differences and as of yet undefined oncogenic drivers of these particular types of breast cancer. Methods: 4 independent datasets were used to correlate gene expression with local recurrence (LR) after 3 years. Kaplan-Meier analysis was used to validate the impact of TTK expression on LR. The TCGA and institutional breast cancer dataset were used to determine TTK expression in BC subtypes. TTK RNA and protein levels were measured using qPCR and western blot at baseline and correlated to intrinsic radiosensitivity. Clonogenic survival assays were used to determine the radiosensitization of cell lines after TTK inhibition (TTKi). Mice models were used to assess TTKi in combination with RT in vivo. DNA damage was quantified using γH2AX staining. HR and NHEJ efficiency assays were performed using HR/NHEJ specific reporter systems. HR competency was also assessed using RAD51 foci formation assays. Results: Ten genes were found to significantly correlate with early LR (≤3 years) across 4 distinct datasets (N=896 pts) (OR of recurrence &amp;gt; 2, p-value &amp;lt;0.000001), with TTK, a cell cycle kinase, ranked the highest. Kaplan-Meier survival analysis in 3 cohorts demonstrated that higher than median TTK expression correlates with decrease LR free survival after RT (HR 1.70-2.42, p&amp;lt;0.01 for all 3 cohorts). Subtype association analysis demonstrated that TTK expression was most elevated in basal-like breast cancer (p&amp;lt;0.0001). Clonogenic survival assays, using inducible shRNA models, show the combination of TTK knockdown and RT increases radiosensitivity in MDA-MB-231 and BT-549 basal-like breast cancer cell lines (rER 1.21-1.63). Additionally, TTKi using, Bayer 1161909 (B909), enhanced radiosensitivity in both cell lines (rER 1.10-1.39). In vivo, TTKi, using shRNA and B909 in combination with RT led to a significant increased time to tumor tripling. Using γH2AX foci formation assays, increased DNA damage was found after combination treatment of TTKi and RT compared to RT alone, indicating that DNA damage repair mechanisms may be compromised by TTKi. We found that the efficiency of the double strand DNA damage repair mechanism, homologous recombination (HR), but not non-homologous end joining (NHEJ), was reduced upon TTKi in HR/NHEJ specific reporter systems. Additionally, RAD51 foci formation was reduced by TTKi after RT compared to RT alone. Conclusion: These data support TTK inhibition as a rationale radiosensitizing strategy for clinical development in basal-like breast cancer patients and that the mechanism of radiosensitization is, at least in part, through impaired HR repair. Citation Format: Benjamin C. Chandler, Leah Moubadder, Cassie Ritter, Kari Wilder-Romans, Meleah Cameron, Meilan Liu, Shyam Nyati, Andrea Pesch, Anna Michmerhuizen, Eric Olsen, Yashar Niknafs, Arul Chinnaiyan, Corey Speers. Nomination and characterization of TTK for radiosensitization in basal-like breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2926.

Abstract 4630: In vivo characterization of the Duffy antigen receptor for chemokines (DARC/ACKR1) in breast cancer tumor progression

Abstract In studies of the tumor microenvironment (TME), factors that influence immune cell infiltration are of great interest, as these populations can influence disease prognosis, and potential treatment for patients. Through in silico analysis of TCGA breast cancer (BC) cohort data, we have identified the Duffy antigen receptor for chemokine/atypical chemokine receptor 1 (DARC/ACKR1) as a potential driver of immune cell infiltration in the BC TME. DARC/ACKR1 is an atypical chemokine receptor that modulates levels of chemokine both in circulation through expression on red blood cells, and participates in chemokine transcytosis in tissues through expression on both post-capillary venules of endothelial cells and epithelial cells. As chemokines establish gradients to recruit specific immune cell types, we hypothesize that the function of DARC/ACKR1 in chemokine level modulation also influences tumor-associated immune cell levels. In the TCGA BC cohort, we observe a strongly positive and significant correlation between DARC/ACKR1 expression and total number of tumor-associated leukocytes, specifically populations of B cells, T cell, monocytes and macrophages. To further characterize the role of DARC/ACKR1 in vivo, we have established a novel DARC knock-out BC transgenic mouse model to study DARC/ACKR1 in the BC TME. To develop our target experimental mice, Ackr1-/- female mice were crossed with Ackr1 +/-; C3(1)Tag +/0 to generate the target Ackr1 -/-; C3(1)Tag +/0 experimental mice. Experimental mice and the littermate controls were aged to 3.5 months, at which point they were euthanized. Blood was collected from the mice to characterize the circulating chemokine profile. Mammary glands containing tumors were fixed and paraffin embedded, followed by IHC and immunofluorescent staining for expression of DARC/ACKR1, target chemokines, immune cells, epithelial cells and endothelial cells. The remaining glands were dissociated, followed by fluorescent-activating cell sorting analysis (FACS) to quantify abundance of immune cells in the gland. Using these methods, we have defined the circulating chemokine profile alongside patterns of immune cell infiltration in our target DARC/ACKR1 knockout BC mice compared to littermate controls in mouse mammary tumors. Citation Format: Rachel N. Martini, Brittany D. Jenkins, Lisa A. Newman, Nancy Manley, Melissa B. Davis. In vivo characterization of the Duffy antigen receptor for chemokines (DARC/ACKR1) in breast cancer tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4630.