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Purpose:We previously demonstrated that high levels of circulating methylated DNA are associated with subsequent disease progression in women with metastatic breast cancer (MBC). In this study, we evaluated the clinical utility of a novel liquid biopsy-breast cancer methylation (LBx-BCM) prototype assay using the GeneXpert cartridge system for early assessment of disease progression in MBC.Experimental Design:The 9-marker LBx-BCM prototype assay was evaluated in TBCRC 005, a prospective biomarker study, using plasma collected at baseline, week 4, and week 8 from 144 patients with MBC.Results:At week 4, patients with MBC with high cumulative methylation (CM) had a significantly shorter median PFS (2.88 months vs. 6.60 months, P = 0.001) and OS (14.52 months vs. 22.44 months, P = 0.005) compared with those with low CM. In a multivariable model, high versus low CM was also associated with shorter PFS (HR, 1.90; 95% CI, 1.20–3.01; P = 0.006). Change in CM from baseline to week 4 (OR, 4.60; 95% CI, 1.77–11.93; P = 0.002) and high levels of CM at week 4 (OR, 2.78; 95% CI, 1.29–5.99; P = 0.009) were associated with progressive disease at the time of first restaging. A robust risk model based on week 4 circulating CM levels was developed to predict disease progression as early as 3 months after initiating a new treatment.Conclusions:The automated LBx-BCM prototype assay is a promising clinical tool for detecting disease progression a month after initiating treatment in women with MBC undergoing routine care. The next step is to validate its clinical utility for specific treatments.

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.

Jianhua Liu,1,* Liantao Guo,1,* Yan Rao,2 Weijie Zheng,1 Dongcheng Gao,1 Jing Zhang,2 Lan Luo,1 Xinwen Kuang,1 Saraswati Sukumar,3 Yi Tu,1 Chuang Chen,1 Shengrong Sun1 1Department of Breast and Thyroid Surgery, Renmin Hospital of Wuhan University, Wuhan, People’s Republic of China; 2Animal Biosafety Level III Laboratory at the Center for Animal Experiment, Wuhan University School of Medicine, Wuhan, People’s Republic of China; 3Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA*These authors contributed equally to this workCorrespondence: Shengrong Sun; Chuang Chen, Tel +86-13707198696 ; +86-1476064785, Email sun137@sina.com; chenc2469@163.comPurpose: Triple-negative breast cancer (TNBC) is challenging to treat with traditional “standard of care” therapy due to the lack of targetable biomarkers and rapid progression to distant metastasis.Methods: We synthesized a novel combination regimen that included chemotherapy and photothermal therapy (PTT) to address this problem. Here, we tested a magnetic nanosystem (MNs-PEG/IR780-DOX micelles) loaded with the near-infrared (NIR) photothermal agent IR780 and doxorubicin (DOX) to achieve chemo-photothermal and boost antitumor immunity. Intraductal (i.duc) administration of MNs-PEG/IR780-DOX could increase the concentration of the drug in the tumor while reducing systemic side effects.Results: We showed more uptake of MNs-PEG/IR780-DOX by 4T1-luc cells and higher penetration in the tumor. MNs-PEG/IR780-DOX exhibited excellent photothermal conversion in vivo and in vitro. The release of DOX from MNs-PEG/IR780-DOX is pH- and temperature-sensitive. Facilitated by i.duc administration, MNs-PEG/IR780-DOX displayed antitumor effects and prevented distant organs metastasis under NIR laser (L) irradiation and magnetic field (MF)while avoiding DOX-induced toxicity. More importantly, MNs-PEG/IR780-DOX alleviated tumor immunosuppressive microenvironment by increasing tumor CD8+ T cells infiltration and reducing the proportion of myeloid-derived suppressor cells (MDSCs) and Tregs.Conclusion: Intraductal administration of pH- and temperature-sensitive MNs-PEG/IR780-DOX with L and MF had the potential for achieving minimally invasive, targeted, and accurate treatment of TNBC.Keywords: triple-negative breast cancer, photothermal therapy, near-infrared image, intraductal administration, tumor immunosuppressive microenvironment

Current molecular liquid biopsy assays to detect recurrence or monitor response to treatment require sophisticated technology, highly trained personnel, and a turnaround time of weeks. We describe the development and technical validation of an automated Liquid Biopsy for Breast Cancer Methylation (LBx-BCM) prototype, a DNA methylation detection cartridge assay that is simple to perform and quantitatively detects nine methylated markers within 4.5 hours. LBx-BCM demonstrated high interassay reproducibility when analyzing exogenous methylated DNA (75–300 DNA copies) spiked into plasma (coefficient of variation, CV = 7.1%–10.9%) and serum (CV = 19.1%–36.1%). It also demonstrated high interuser reproducibility (Spearman r = 0.887, P < 0.0001) when samples of metastatic breast cancer (MBC, N = 11) and normal control (N = 4) were evaluated independently by two users. Analyses of interplatform reproducibility indicated very high concordance between LBx-BCM and the reference assay, cMethDNA, among 66 paired plasma samples [MBC N = 40, controls N = 26; Spearman r = 0.891; 95% confidence interval (CI) = 0.825–0.933, P < 0.0001]. LBx-BCM achieved a ROC AUC = 0.909 (95% CI = 0.836–0.982), 83% sensitivity and 92% specificity; cMethDNA achieved a ROC AUC = 0.896 (95% CI = 0.817–0.974), 83% sensitivity and 92% specificity in test set samples. The automated LBx-BCM cartridge prototype is fast, with performance levels equivalent to the highly sensitive, manual cMethDNA method. Future prospective clinical studies will evaluate LBx-BCM detection sensitivity and its ability to monitor therapeutic response during treatment for advanced breast cancer. Significance: We technically validated an automated, cartridge-based, liquid biopsy prototype assay, to quantitatively measure breast cancer methylation in serum or plasma of patients with MBC, that demonstrated high sensitivity and specificity.

The table provides a step by step guide to calculate methylation in each gene followed by cumulative methylation using the algorithm derived in this paper.

Figure shows the perfomance of LBx-BCM in the training set samples. A histogram shows cumulative methylation for each sample, and a box plot shows significnt difference of methylation in serum of metastatic breast cancer patients compared to normal individuals (Mann Whitney p= 0.002)

Current molecular liquid biopsy assays to detect recurrence or monitor response to treatment require sophisticated technology, highly trained personnel, and a turnaround time of weeks. We describe the development and technical validation of an automated Liquid Biopsy for Breast Cancer Methylation (LBx-BCM) prototype, a DNA methylation detection cartridge assay that is simple to perform and quantitatively detects nine methylated markers within 4.5 hours. LBx-BCM demonstrated high interassay reproducibility when analyzing exogenous methylated DNA (75–300 DNA copies) spiked into plasma (coefficient of variation, CV = 7.1%–10.9%) and serum (CV = 19.1%–36.1%). It also demonstrated high interuser reproducibility (Spearman r = 0.887, P < 0.0001) when samples of metastatic breast cancer (MBC, N = 11) and normal control (N = 4) were evaluated independently by two users. Analyses of interplatform reproducibility indicated very high concordance between LBx-BCM and the reference assay, cMethDNA, among 66 paired plasma samples [MBC N = 40, controls N = 26; Spearman r = 0.891; 95% confidence interval (CI) = 0.825–0.933, P < 0.0001]. LBx-BCM achieved a ROC AUC = 0.909 (95% CI = 0.836–0.982), 83% sensitivity and 92% specificity; cMethDNA achieved a ROC AUC = 0.896 (95% CI = 0.817–0.974), 83% sensitivity and 92% specificity in test set samples. The automated LBx-BCM cartridge prototype is fast, with performance levels equivalent to the highly sensitive, manual cMethDNA method. Future prospective clinical studies will evaluate LBx-BCM detection sensitivity and its ability to monitor therapeutic response during treatment for advanced breast cancer.Significance:We technically validated an automated, cartridge-based, liquid biopsy prototype assay, to quantitatively measure breast cancer methylation in serum or plasma of patients with MBC, that demonstrated high sensitivity and specificity.

Table S1: DNA methylation in rFNA samples Table S2: Association between methylation and menopause as measured by LH and FSH- Univariate analysis Table S3: Association between Methylation and Menstrual Cycle as Measured by Estradiol and Progesterone - Univariate Analysis Table S4: Association between DNA methylation and lifetime Gail risk score - multivariate analysis Table S5: Association between DNA Methylation and percent breast density Table S6: Association between DNA Methylation and Masood cytology score Table S7: Gene methylation versus standard cytology Table S8: Spearman correlation: relationship between biomarkers within the panel Table S9: Agreement between baseline and 6 month methylation levels in rFNA among individuals (n=15).

Sex steroid hormones contribute to breast cancer development, but data on concentrations of these within breast tissue are limited. We performed simultaneous multiparameter measurement of breast sex steroids, breast epithelial cytology, and DNA methylation in 119 healthy women (54 pre- and 65 postmenopausal) without a history of breast cancer. Random fine-needle aspiration (rFNA) of the breast was performed simultaneously with blood collection. Breast samples were analyzed by LC/MS-MS for estrone, estradiol, progesterone, androstenedione, and testosterone. Blood samples were assayed for estradiol and progesterone by immunoassay. Cytomorphology was classified using the Masood Score, and DNA methylation of eight genes was analyzed using quantitative multiplexed methylation-specific PCR, and expressed as the cumulative methylation index (CMI). Serum and breast concentrations of estradiol and progesterone showed significant correlation (Spearman r = 0.34, Padj = 0.001 and r = 0.69, Padj < 0.0006, respectively). Progesterone concentration was significantly higher in the premenopausal breast (Padj < 0.0008), and showed a luteal surge. Breast estrone and estradiol concentrations did not differ significantly by menopause, but androstenedione concentration was higher in the breasts of postmenopausal women (P = 0.026 and Padj = 0.208). Breast androgens were significantly correlated with breast density (Spearman r = 0.27, Padj = 0.02 for testosterone) and CMI (Spearman r = 0.3, Padj = 0.038 for androstenedione). Our data indicate that future larger studies of breast steroid hormones along with other parameters are feasible. Significant associations of breast androgen concentrations with breast density and gene methylation warrant future study. Cancer Prev Res; 11(9); 557–68. ©2018 AACR.

Table S1. Correlations between parameters that influence the quality of the rFNA sample (119 women) Table S2. Correlation between Breast and serum hormone concentrations. Table S3. Cytology according to menopausal status and menstrual cycle.

Peptide therapeutics hold great promise for the treatment of cancer due to low toxicity, high specificity, and ease of synthesis and modification. However, the unfavorable pharmacokinetic parameters strictly limit their therapeutic efficacy and clinical translation. Here, we tailor-designed an amphiphilic chimeric peptide through conjugation of functional 3-diethylaminopropyl isothiocyanate (DEAP) molecules to a short antitumor peptide, C16Y. The ultimate DEAP–C16Y peptides self-assembled into spherical nanostructures at physiologic conditions, which dissociated to release individual peptide molecules in weakly acidic tumors. DEAP–C16Y peptides showed negligible cytotoxicity but impaired vascular endothelial cell migration and tubule formation by inactivation of the focal adhesion kinase and PI3K–Akt pathways, as well as tumor cell invasion by decreasing invadopodia formation. Compared with C16Y, the systemically administered DEAP–C16Y nanostructures exhibited superior stability, thereby allowing prolonged treatment interval and resulting in significant decreases in microvessel density, tumor growth, and distant metastasis formation in orthotopic mammary tumor models. Through encapsulation of hydrophobic doxorubicin, DEAP–C16Y nanostructure served as a smart carrier to achieve targeted drug delivery and combination therapy. Our study, for the first time, demonstrates that a simple nanoformulation using a functional antitumor peptide as the building block can show innate antitumor activity and also provide a nanoplatform for combination therapy, opening a new avenue for the design of antitumor nanotherapeutics. Mol Cancer Ther; 14(10); 2390–400. ©2015 AACR.

Supplementary Materials and Methods; Supplementary Table S1; Supplementary Figures S1-S5. Supplementary materials and methods shows detailed materials and methods of evaluation of the drug loading and release profiles of DEAP-C16Y-Dox. Supplementary Table S1 shows encapsulation and loading efficiency of Dox in DEAP-C16Y-Dox or DEAP-HSAF-Dox. Supplementary Figure S1 shows characterization of the DEAP-C16Y self-assembling nanoprobe. Supplementary Figure S2 shows DEAP-HSAF nanostructure and effects of DEAP-C16Y on endothelial cells and tumor cells. Supplementary Figure S3 shows biodistribution and antitumor efficacy of DEAP-C16Y nanostructures. Supplementary Figure S4 shows the potential toxicity of DEAP-C16Y nanostructures. Supplementary Figure S5 shows combination therapy of DEAP-C16Y and Dox.

Supplementary Figure S1 (related to Figure 1). HOXB7 microarray and ChIP-PED analysis. Supplementary Figure S2 (related to Figure 1). HOXB7 enhances expression of ER target genes. Supplementary Figure S3 (related to Figure 2). Validation of HOXB7 binding events to ER target genes. Supplementary Figure S4 (related to Figure 2). HOXB7 is required for ER target gene expression. Supplementary Figure S5 (related to Figure 3). Correlation between HOXB7 and HER2 expression. Supplementary Figure S6 (related to Figure 3). HOXB7 regulates HER2 expression. Supplementary Figure S7 (related to Figure 4). miRNA-196a regulates HOXB7 expression. Supplementary Figure S8 (related to Figure 5). MYC and miRNA-196a regulates HOXB7 expression. Supplementary Figure S9 (related to Figure 5). In vivo analysis of effects of inhibiting MYC and HER2 to overcome TAM-resistance in BT474 cells. Supplementary Figure S10 (related to Figure 6). MYC-HOXB7-HER2 predicts clinical outcome of breast cancer in women treated with endocrine therapy. Supplementary Table S1 (related to Figure 1), Family wise error rate (FWER) for MR-GSE of genes differentially expressed between MCF-7-HOXB7 and MCF-7-Vector cells. Supplementary Table S2 (related to Figure 1). ER target genes ranked in order of the average correlation with HOXB7 or ER.

Figure S1: Performance of a minimal 5-gene marker panel.

Related Article from Big Punches Come in Nanosizes for Chemoprevention

Purpose:We previously demonstrated that high levels of circulating methylated DNA are associated with subsequent disease progression in women with metastatic breast cancer (MBC). In this study, we evaluated the clinical utility of a novel liquid biopsy-breast cancer methylation (LBx-BCM) prototype assay using the GeneXpert cartridge system for early assessment of disease progression in MBC.Experimental Design:The 9-marker LBx-BCM prototype assay was evaluated in TBCRC 005, a prospective biomarker study, using plasma collected at baseline, week 4, and week 8 from 144 patients with MBC.Results:At week 4, patients with MBC with high cumulative methylation (CM) had a significantly shorter median PFS (2.88 months vs. 6.60 months, P = 0.001) and OS (14.52 months vs. 22.44 months, P = 0.005) compared with those with low CM. In a multivariable model, high versus low CM was also associated with shorter PFS (HR, 1.90; 95% CI, 1.20–3.01; P = 0.006). Change in CM from baseline to week 4 (OR, 4.60; 95% CI, 1.77–11.93; P = 0.002) and high levels of CM at week 4 (OR, 2.78; 95% CI, 1.29–5.99; P = 0.009) were associated with progressive disease at the time of first restaging. A robust risk model based on week 4 circulating CM levels was developed to predict disease progression as early as 3 months after initiating a new treatment.Conclusions:The automated LBx-BCM prototype assay is a promising clinical tool for detecting disease progression a month after initiating treatment in women with MBC undergoing routine care. The next step is to validate its clinical utility for specific treatments.

Association between week 4 CM and disease status at first restaging after adjusting for CEA.

Supplementary Figure 2 displays the relationship between the week 8 CM level and the change in CM level (Week 8 – baseline). Figure S4B and C use box plots to display the relationship between disease status at first restaging and week 8 CM level or change in CM level (Week 8 – baseline).

Supplementary Data from Heterogeneity of Breast Cancer Metastases: Comparison of Therapeutic Target Expression and Promoter Methylation Between Primary Tumors and Their Multifocal Metastases

PDF file, 67K

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 α, APC1, and RARβ 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.

PDF file, 85K - Supplementary Figure 1 Pre and Post Vorinostat Values of Other Gene Expression using the Oncotype DX 21-gene Assay.

Purpose: Agents that target the epigenome show activity in breast cancer models. In preclinical studies, the histone deacetylase inhibitor vorinostat induces cell-cycle arrest, apoptosis, and differentiation. We evaluated biomarker modulation in breast cancer tissues obtained from women with newly diagnosed invasive disease who received vorinostat and those who did not.Experimental Design: Tumor specimens were collected from 25 women who received up to 6 doses of oral vorinostat 300 mg twice daily and from 25 untreated controls in a nonrandomized study. Candidate gene expression was analyzed by reverse transcription PCR (RT-PCR) using the Oncotype DX 21-gene assay, and by immunohistochemistry for Ki-67 and cleaved caspase-3. Matched samples from treated women were analyzed for gene methylation by quantitative multiplex methylation-specific PCR (QM-MSP). Wilcoxon nonparametric tests were used to compare changes in quantitative gene expression levels pre- and post-vorinostat with changes in expression in untreated controls, and changes in gene methylation between pre- and post-vorinostat samples.Results: Vorinostat was well tolerated and there were no study-related delays in treatment. Compared with untreated controls, there were statistically significant decreases in the expression of proliferation-associated genes Ki-67 (P = 0.003), STK15 (P = 0.005), and Cyclin B1 (P = 0.03) following vorinostat, but not in other genes by the Oncotype DX assay, or in expression of Ki-67 or cleaved caspase-3 by immunohistochemistry. Changes in methylation were not observed.Conclusions: Short-term vorinostat administration is associated with a significant decrease in expression of proliferation-associated genes in untreated breast cancers. This demonstration of biologic activity supports investigation of vorinostat in combination with other agents for the management of breast cancer. Clin Cancer Res; 19(14); 4008–16. ©2013 AACR.

Supplementary methods and figures S1-S8. (S1): Glutamine dependency and induction of apoptosis on glutamine withdrawal in breast cancer cells. (S2): Cytotoxicity of AOA in breast cancer cells. (S3): Myc regulation of transaminases and glutamine dependency in mouse tumor cell lines. (S4): NMR spectra of AOA-treated compared to vehicle-treated SUM159 cells. (S5): Cell cycle arrest and BrdU incorporation after AOA treatment in SUM149 cells; irreversible growth arrest by AOA in SUM149 and 159 cells; partial rescue of cell death due to glutamine withdrawal by exogenous aspartate in breast cancer cell lines. (S6): Western blot analysis showing change in ER stress markers, energy metabolism and apoptosis by AOA treatment in SUM149, SUM159 and MDA-MB-231 cells. (S7): Tumor growth inhibition by AOA in MCF-7, SUM149 and SUM159 xenografts; immunohistochemistry analysis of cleaved caspase 3 in AOA treated tumors; quantitation of ER stress markers in AOA treated tumors. (S8): Effect of AOA in combination with paclitaxel or carboplatin in SUM149 and SUM159 xenografts; up regulation of c-myc in carboplatin and paclitaxel treated MDA-MB-231 xenografts.

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

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. Clin Cancer Res; 21(14); 3263–73. ©2015 AACR.

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.

PDF file, 154K, Table S1: Primers used for PCR ; Table S2. Antibodies used for Western Blot ; Table S3 Treatment comparisons of tumor volumes at selected time points. ; Table S4: Treatment comparisons of tumor volumes at selected time points -MCF7-HOXB13 cell line, SC xenografts ; Table S5: Treatment comparisons of tumor volumes at selected time points MCF7-LPCX cell line, MFP xenografts ; Table S6. Treatment comparisons of tumor volumes at selected time points-MCF7-HOXB13 cell line, MFP xenografts.

Supplementary Figure 4 from SLITs Suppress Tumor Growth In vivo by Silencing Sdf1/Cxcr4 within Breast Epithelium

XLSX file - 32K, Shannon and Simpson's index for each tumor and each probe based on BAC, CEP, BAC/CEP and unique counts.

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. Cancer Res; 74(8); 2160–70. ©2014 AACR.

Supplementary Figure Legends. Legend for Supplementary Figures S1-S10.

Supplemental Materials and Methods and Supplemental References

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