HOXA5-Mediated Stabilization of IκBα Inhibits the NF-κB Pathway and Suppresses Malignant Transformation of Breast Epithelial Cells.

HOXA5 is a transcription factor and tumor suppressor that promotes differentiation of breast epithelial cells and is frequently lost during malignant transformation. HOXA5 loss alone, however, does not confer tumorigenicity. To determine which molecular alterations combined with loss of HOXA5 expression can transform cells, we examined isogenic derivatives of a nonmalignant breast epithelial cell line containing knock-in or knockout mutations in key breast cancer genes. Knockdown (KD) of HOXA5 in cells harboring double knock-in (DKI) of mutated PIK3CA (E545K) and HER2 (V777L) induced epithelial-mesenchymal transition and migration and promoted invasive tumor outgrowth within mouse mammary ducts. The NF-κB pathway was significantly upregulated in DKI cells following HOXA5 KD. HOXA5 KD upregulated multiple NF-κB target genes, including IL6. IκBα protein, but not RNA, expression was reduced in HOXA5-KD cells. HOXA5 bound and stabilized IκBα, forming a nuclear HOXA5-IκBα complex. Chromatin immunoprecipitation sequencing database queries revealed that HOXA5 and IκBα are co-enriched at 528 genomic loci. In patients with breast cancer, high coexpression of HOXA5 and IκBα conferred a significantly better overall and progression-free survival. Collectively, these data suggest that HOXA5 suppresses malignancy in breast epithelial cells by blunting NF-κB action via stabilization of its inhibitor IκBα. SIGNIFICANCE: Loss of HOXA5 reduces IκBα stability and increases NF-κB signaling to exacerbate breast cancer aggressiveness, providing new insights into the tumor suppressor functions of HOXA5.

Non-coding RNA LEVER sequestration of PRC2 can mediate long range gene regulation.

Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator required for gene silencing during development. Although PRC2 is a well-established RNA-binding complex, the biological function of PRC2-RNA interaction has been controversial. Here, we study the gene-regulatory role of the inhibitory PRC2-RNA interactions. We report a nuclear long non-coding RNA, LEVER, which mapped 236 kb upstream of the ß-globin cluster as confirmed by Nanopore sequencing. LEVER RNA interacts with PRC2 in its nascent form, and this prevents the accumulation of the H3K27 repressive histone marks within LEVER locus. Interestingly, the accessible LEVER chromatin, in turn, suppresses the chromatin interactions between the ε-globin locus and ß-globin locus control region (LCR), resulting in a repressive effect on ε-globin gene expression. Our findings validate that the nascent RNA-PRC2 interaction inhibits local PRC2 function in situ. More importantly, we demonstrate that such a local process can in turn regulate the expression of neighboring genes.

HEYL Regulates Neoangiogenesis Through Overexpression in Both Breast Tumor Epithelium and Endothelium.

Blocking tumor angiogenesis is an appealing therapeutic strategy, but to date, success has been elusive. We previously identified HEYL, a downstream target of Notch signaling, as an overexpressed gene in both breast cancer cells and as a tumor endothelial marker, suggesting that HEYL overexpression in both compartments may contribute to neoangiogenesis. Carcinomas arising in double transgenic Her2-neu/HeyL mice showed higher tumor vessel density and significantly faster growth than tumors in parental Her2/neu mice. Providing mechanistic insight, microarray-based mRNA profiling of HS578T-tet-off-HEYL human breast cancer cells revealed upregulation of several angiogenic factors including CXCL1/2/3 upon HEYL expression, which was validated by RT-qPCR and protein array analysis. Upregulation of the cytokines CXCL1/2/3 occurred through direct binding of HEYL to their promoter sequences. We found that vessel growth and migration of human vascular endothelial cells (HUVECs) was promoted by conditioned medium from HS578T-tet-off-HEYL carcinoma cells, but was blocked by neutralizing antibodies against CXCL1/2/3. Supporting these findings, suppressing HEYL expression using shRNA in MDA-MB-231 cells significantly reduced tumor growth. In addition, suppressing the action of proangiogenic cytokines induced by HEYL using a small molecule inhibitor of the CXCl1/2/3 receptor, CXCR2, in combination with the anti-VEGF monoclonal antibody, bevacizumab, significantly reduced tumor growth of MDA-MB-231 xenografts. Thus, HEYL expression in tumor epithelium has a profound effect on the vascular microenvironment in promoting neoangiogenesis. Furthermore, we show that lack of HEYL expression in endothelial cells leads to defects in neoangiogenesis, both under normal physiological conditions and in cancer. Thus, HeyL-/- mice showed impaired vessel outgrowth in the neonatal retina, while the growth of mammary tumor cells E0771 was retarded in syngeneic HeyL-/- mice compared to wild type C57/Bl6 mice. Blocking HEYL's angiogenesis-promoting function in both tumor cells and tumor-associated endothelium may enhance efficacy of therapy targeting the tumor vasculature in breast cancer.

Tibial fixation in anterior cruciate ligament reconstruction.

OBJECTIVE: To determine whether supplementary tibial graft fixation with a staple is routinely necessary for anterior cruciate ligament (ACL) reconstructions. METHODS: We retrospectively reviewed a series of consecutive patients who underwent ACL reconstruction at our institution from April 2012 to July 2013. Patients who fulfilled the inclusion and exclusion criteria were divided into two groups, of which one with tibial fixation of the graft with a biointerference screw alone (biointerference screw group) and the other with tibial fixation of the graft with a biointerference screw and supplementary extra tunnel staple fixation (biointerference screw and staple group). All the surgeries were performed by a single fellowship-trained sports surgeon, using a standardized transportal technique and rehabilitation protocol. Both well-matched groups were evaluated at 1-year follow-up objectively for ligament laxity using instrumented testing with KT-2000 arthrometer and clinical tests as well as subjectively with the validated International Knee Documentation Committee 2000 (IKDC) and Lysholm knee score. All complications were reported. RESULTS: A total of 64 patients (31 in the only biointerference screw group and 33 in the biointerference screw and staple group) were included in the study. At 1 year, there was no significant difference in the objective and subjective outcome assessments between the two groups. However, four patients from the group with supplementary staple experienced symptomatic hardware on kneeling of which two necessitated removal of hardware. CONCLUSION: Our study showed that supplementary tibial graft fixation with a staple is not routinely necessary for ACL reconstructions. It confers no additional benefits when compared with the use of biointerference screw alone for tibial graft fixation but may increase the risks of symptomatic hardware.

HOXC10 Expression Supports the Development of Chemotherapy Resistance by Fine Tuning DNA Repair in Breast Cancer Cells.

Development of drug resistance is a major factor limiting the continued success of cancer chemotherapy. To overcome drug resistance, understanding the underlying mechanism(s) is essential. We found that HOXC10 is overexpressed in primary carcinomas of the breast, and even more significantly in distant metastasis arising after failed chemotherapy. High HOXC10 expression correlates with shorter recurrence-free and overall survival in patients with estrogen receptor-negative breast cancer undergoing chemotherapy. We found that HOXC10 promotes survival in cells treated with doxorubicin, paclitaxel, or carboplatin by suppressing apoptosis and upregulating NF-κB Overexpressed HOXC10 increases S-phase-specific DNA damage repair by homologous recombination (HR) and checkpoint recovery in cells at three important phases. For double-strand break repair, HOXC10 recruits HR proteins at sites of DNA damage. It enhances resection and lastly, it resolves stalled replication forks, leading to initiation of DNA replication following DNA damage. We show that HOXC10 facilitates, but is not directly involved in DNA damage repair mediated by HR. HOXC10 achieves integration of these functions by binding to, and activating cyclin-dependent kinase, CDK7, which regulates transcription by phosphorylating the carboxy-terminal domain of RNA polymerase II. Consistent with these findings, inhibitors of CDK7 reverse HOXC10-mediated drug resistance in cultured cells. Blocking HOXC10 function, therefore, presents a promising new strategy to overcome chemotherapy resistance in breast cancer. Cancer Res; 76(15); 4443-56. ©2016 AACR.

HOXB7 Is an ERα Cofactor in the Activation of HER2 and Multiple ER Target Genes Leading to Endocrine Resistance.

UNLABELLED: Why breast cancers become resistant to tamoxifen despite continued expression of the estrogen receptor-α (ERα) and what factors are responsible for high HER2 expression in these tumors remains an enigma. HOXB7 chromatin immunoprecipitation analysis followed by validation showed that HOXB7 physically interacts with ERα, and that the HOXB7-ERα complex enhances transcription of many ERα target genes, including HER2. Investigating strategies for controlling HOXB7, our studies revealed that MYC, stabilized via phosphorylation mediated by EGFR-HER2 signaling, inhibits transcription of miR-196a, a HOXB7 repressor. This leads to increased expression of HOXB7, ER target genes, and HER2. Repressing MYC using small-molecule inhibitors reverses these events and causes regression of breast cancer xenografts. The MYC-HOXB7-HER2 signaling pathway is eminently targetable in endocrine-resistant breast cancer. SIGNIFICANCE: HOXB7 acts as an ERα cofactor regulating a myriad of ER target genes, including HER2, in tamoxifen-resistant breast cancer. HOXB7 expression is controlled by MYC via transcriptional regulation of the HOXB7 repressor miR-196a; consequently, antagonists of MYC cause reversal of selective ER modulator resistance both in vitro and in vivo.

Targeting Glutamine Metabolism in Breast Cancer with Aminooxyacetate.

PURPOSE: Glutamine addiction in c-MYC-overexpressing breast cancer is targeted by the aminotransferase inhibitor, aminooxyacetate (AOA). However, the mechanism of ensuing cell death remains unresolved. EXPERIMENTAL DESIGN: A correlation between glutamine dependence for growth and c-MYC expression was studied in breast cancer cell lines. The cytotoxic effects of AOA, its correlation with high c-MYC expression, and effects on enzymes in the glutaminolytic pathway were investigated. AOA-induced cell death was assessed by measuring changes in metabolite levels by magnetic resonance spectroscopy (MRS), the effects of amino acid depletion on nucleotide synthesis by cell-cycle and bromodeoxyuridine (BrdUrd) uptake analysis, and activation of the endoplasmic reticulum (ER) stress-mediated pathway. Antitumor effects of AOA with or without common chemotherapies were determined in breast cancer xenografts in immunodeficient mice and in a transgenic MMTV-rTtA-TetO-myc mouse mammary tumor model. RESULTS: We established a direct correlation between c-MYC overexpression, suppression of glutaminolysis, and AOA sensitivity in most breast cancer cells. MRS, cell-cycle analysis, and BrdUrd uptake measurements indicated depletion of aspartic acid and alanine leading to cell-cycle arrest at S-phase by AOA. Activation of components of the ER stress-mediated pathway, initiated through GRP78, led to apoptotic cell death. AOA inhibited growth of SUM159, SUM149, and MCF-7 xenografts and c-myc-overexpressing transgenic mouse mammary tumors. In MDA-MB-231, AOA was effective only in combination with chemotherapy. CONCLUSIONS: AOA mediates its cytotoxic effects largely through the stress response pathway. The preclinical data of AOA's effectiveness provide a strong rationale for further clinical development, particularly for c-MYC-overexpressing breast cancers.

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.

Combining the strength of genomics, nanoparticle technology, and direct intraductal delivery for breast cancer treatment.

A large number of genes are altered in cancer cells. Often, reversal or inhibition of just one of these alterations leads to death of the cancer cells. Technological advances in multiple areas are necessary to potentiate clinical translation of these findings. In a recent article, Brock and colleagues reported that overexpressed HOXA1 is a critical event in tumor progression in a mouse mammary tumor model. They developed HOXA1-small interfering RNA nanoparticles and achieved effective therapeutic doses by delivering them intraductally through the nipple to the site of the tumor and at the same time circumvented the systemic immune response. This study strengthens the concept of targeting overexpressed genes by using small interfering RNA and bypassing systemic immunity through local intraductal delivery.

Genome-wide methylation analysis identifies genes specific to breast cancer hormone receptor status and risk of recurrence.

To better understand the biology of hormone receptor-positive and-negative breast cancer and to identify methylated gene markers of disease progression, we carried out a genome-wide methylation array analysis on 103 primary invasive breast cancers and 21 normal breast samples, using the Illumina Infinium HumanMethylation27 array that queried 27,578 CpG loci. Estrogen and/or progesterone receptor-positive tumors displayed more hypermethylated loci than estrogen receptor (ER)-negative tumors. However, the hypermethylated loci in ER-negative tumors were clustered closer to the transcriptional start site compared with ER-positive tumors. An ER-classifier set of CpG loci was identified, which independently partitioned primary tumors into ER subtypes. A total of 40 (32 novel and 8 previously known) CpG loci showed differential methylation specific to either ER-positive or ER-negative tumors. Each of the 40 ER subtype-specific loci was validated in silico, using an independent, publicly available methylome dataset from the Cancer Genome Atlas. In addition, we identified 100 methylated CpG loci that were significantly associated with disease progression; the majority of these loci were informative particularly in ER-negative breast cancer. Overall, the set was highly enriched in homeobox containing genes. This pilot study shows the robustness of the breast cancer methylome and illustrates its potential to stratify and reveal biological differences between ER subtypes of breast cancer. Furthermore, it defines candidate ER-specific markers and identifies potential markers predictive of outcome within ER subgroups.

MethySYBR, a novel quantitative PCR assay for the dual analysis of DNA methylation and CpG methylation density.

Development of facile, sensitive, specific, and economical assays for the analysis of methylated alleles is crucial to the use of methylated biomarkers for cancer detection. We hereby report a novel method, MethySYBR, a SYBR green-based PCR assay for the dual analysis of DNA methylation and CpG methylation density. MethySYBR begins with multiplex PCR to enable the simultaneous amplification of many discrete target alleles in a single reaction using as little as 3 pg of bisulfite-converted DNA. In the second round of PCR, the specific methylated target is quantified from multiplex products using both nested methylation-independent and methylation-specific primer sets. Moreover, the use of SYBR green dye during quantitative PCR enables melting curve analysis of target amplicons to determine the methylation density of CpG sites on target alleles. To establish proof of principle, two cancer-specific methylated genes, RASSF1A and OGDHL, were assessed by MethySYBR. We demonstrated that MethySYBR sensitively detected methylated alleles in the presence of a 100,000-fold excess of unmethylated allele. Furthermore, MethySYBR was shown to be capable of analyzing minute amounts of DNA from paraffin-embedded tissue. Therefore, the MethySYBR assay is a simple, highly specific, highly sensitive, high-throughput, and cost-effective method that is widely applicable to basic and clinical studies of DNA methylation.

Hypermethylated genes as biomarkers of cancer in women with pathologic nipple discharge.

PURPOSE: In a pilot study of women with pathologic nipple discharge (PND) undergoing ductoscopy, we tested quantitative assessment of gene promoter hypermethylation using quantitative multiplex methylation-specific PCR (QM-MSP) to enhance detection of duct carcinoma in situ (DCIS). EXPERIMENTAL DESIGN: Women with PND underwent ductoscopy; ducts with significant lesions were surgically resected (36 ducts in 33 women) and those with minimal findings were not (28 ducts in 16 women). QM-MSP was done on ductoscopy cell samples. Results were compared with cytology and tissue histology. RESULTS: Cells from ducts with significant lesions on ductoscopy had significantly higher levels of methylation than those with minimal findings. Furthermore, cells from ducts with DCIS displayed higher levels of methylation than those with benign lesions such as papilloma (P = 0.006); or ducts with minimal findings on ductoscopy (P = 0.0001). Cumulative RASSF1A, TWIST1, and HIN1 gene methylation accurately distinguished ducts with cancerous versus benign lesions (100% sensitivity, 72% specificity, and area under the curve of 0.91 according to receiving operating characteristic analyses). QM-MSP analysis was more informative than cytology (100% versus 29% sensitivity, respectively), for detecting DCIS. In a validation set of paraffin-embedded DCIS and papilloma samples from women presenting with PND, QM-MSP was significantly higher in DNA from DCIS than papilloma sections (P = 0.002). CONCLUSION: The positive predictive value of ductoscopy was more than doubled (19% versus 47%) with the addition of QM-MSP, demonstrating the benefit of targeting ducts having both high methylation and significant abnormalities on ductoscopy for surgical excision. Future large-scale studies to validate this approach are needed.

Quantitative promoter hypermethylation profiles of ductal carcinoma in situ in North American and Korean women: Potential applications for diagnosis.

To investigate the diagnostic potential of DNA methylation-based markers in tissue samples of DCIS, we examined the prevalence and extent of methylation in breast ductal carcinoma in situ (DCIS) samples from North American and Korean women. Quantitative multiplex-methylation specific PCR (QM-MSP) of ten genes was performed. The methylation level of APC1, Cyclin D2, HIN-1, RASSF1A and Twist singly, and cumulative methylation of all ten genes was significantly higher in DCIS compared to normal tissues for both groups. A three-gene panel (APC1, HIN-1 and RASSF1A) QM-MSP distinguished between DCIS and normal breast tissues with a sensitivity of 94 to 96% and a specificity of 81 to 87%. Methylation levels of these three genes in DCIS were higher than those of hyperplasia or adjacent normal appearing tissues in Korean women. Comparing North American and Korean DCIS, statistically significant differences in methylation levels were found for CDH1, ERalpha and RAR-beta. Quantification of gene methylation combined with immunohistochemistry in a small subset of tumors suggested that methylation may precede loss of protein expression for ERalpha. Our study demonstrated that methylation profiles of DCIS between North American and Korean women were similar. Methylation status of a panel of genes measured in a quantitative manner accurately discriminated between normal and DCIS tissues of both groups. For both North American and Korean women, QM-MSP analysis of a key panel of genes may be useful as an ancillary tool for DCIS detection in breast tissues.

Heterogeneity of breast cancer metastases: comparison of therapeutic target expression and promoter methylation between primary tumors and their multifocal metastases.

PURPOSE: A comprehensive comparison of biomarker expression between patients' primary breast carcinoma (PBC) and their metastatic breast carcinomas (MBC) has not been done. EXPERIMENTAL DESIGN: We did rapid autopsies (postmortem intervals, 1-4 hours) on 10 consenting patients who died of MBC. We constructed single-patient tissue microarrays from the patients' archived PBC and multiple different MBCs harvested at autopsy, which were immunohistochemically labeled for multiple biomarkers. Methylation of multiple gene promoters was assessed quantitatively on dissected PBC and MBC samples. RESULTS: Extensive heterogeneity was observed between PBC and their paired MBC, as well as among multiple MBC from the same patient. Estrogen and progesterone receptors tended to be uniformly down-regulated in metastases. E-cadherin was down-regulated in a subset of the MBC of one case. Variable overexpression in MBC compared with the PBC was observed for cyclooxygenase-2 (five cases), epidermal growth factor receptor (EGFR; four cases), MET (four cases), and mesothelin (four cases). No case strongly overexpressed HER-2/neu by immunohistochemistry, but eight cases showed variable protein expression ranging from negative to equivocal (2+) in different MBC. In one case, variable low-level HER-2/neu gene amplification was found. EGFR and MET overexpression were restricted to the four basal-type cancers. EGFR protein overexpression did not correlate with EGFR gene amplification. Multigene promoter hypermethylation of RASSF1a, HIN1, cyclin D2, Twist, estrogen receptor alpha, APC1, and RARbeta was overall very similar in the PBC and all MBCs in all cases. CONCLUSIONS: Therapeutic targets identified in the PBC or even some MBC may not reflect targets present in all metastatic sites.