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Pharmacological induction of oxidative and proteotoxic stress has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Guided by a differential phenotypic drug screen for novel lead compounds that selectively induce melanoma cell apoptosis without compromising viability of primary human melanocytes, we have focused on the cyclic pyridinyl-polythiazolyl peptide-antimicrobial thiostrepton. Using comparative gene expression-array analysis, the early cellular stress response induced by thiostrepton was examined in human A375 metastatic melanoma cells and primary melanocytes. Thiostrepton displayed selective antimelanoma activity causing early induction of proteotoxic stress with massive upregulation of heat shock (HSPA6, HSPA1A, DNAJB4, HSPB1, HSPH1, HSPA1L, CRYAB, HSPA5, DNAJA1), oxidative stress (HMOX1, GSR, SOD1), and ER stress response (DDIT3) gene expression, confirmed by immunodetection (Hsp70, Hsp70B', HO-1, phospho-eIF2α). Moreover, upregulation of p53, proapoptotic modulation of Bcl-2 family members (Bax, Noxa, Mcl-1, Bcl-2), and induction of apoptotic cell death were observed. Thiostrepton rapidly induced cellular oxidative stress followed by inactivation of chymotrypsin-like proteasomal activity and melanoma cell-directed accumulation of ubiquitinated proteins, not observed in melanocytes that were resistant to thiostrepton-induced apoptosis. Proteotoxic and apoptogenic effects were fully antagonized by antioxidant intervention. In RPMI 8226 multiple myeloma cells, known to be exquisitely sensitive to proteasome inhibition, early proteotoxic and apoptogenic effects of thiostrepton were confirmed by array analysis indicating pronounced upregulation of heat shock response gene expression. Our findings demonstrate that thiostrepton displays dual activity as a selective prooxidant and proteotoxic chemotherapeutic, suggesting feasibility of experimental intervention targeting metastatic melanoma and other malignancies including multiple myeloma.

Introduction Desmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens. Methods We used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines. Results DSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure. Conclusion These results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure.

DNA methylation plays a role in a variety of biological processes including embryonic development, imprinting, X-chromosome inactivation, and stem cell differentiation. Tissue specific differential methylation has also been well characterized. We sought to extend these studies to create a map of differential DNA methylation between different cell types derived from a single tissue. Using three pairs of isogenic human mammary epithelial and fibroblast cells, promoter region DNA methylation was characterized using MeDIP coupled to microarray analysis. Comparison of DNA methylation between these cell types revealed nearly three thousand cell-type specific differentially methylated regions (ctDMRs). MassARRAY was performed upon 87 ctDMRs to confirm and quantify differential DNA methylation. Each of the examined regions exhibited statistically significant differences ranging from 10-70%. Gene ontology analysis revealed the overrepresentation of many transcription factors involved in developmental processes. Additionally, we have shown that ctDMRs are associated with histone related epigenetic marks and are often aberrantly methylated in breast cancer. Overall, our data suggest that there are thousands of ctDMRs which consistently exhibit differential DNA methylation and may underlie cell type specificity in human breast tissue. In addition, we describe the pathways affected by these differences and provide insight into the molecular mechanisms and physiological overlap between normal cellular differentiation and breast carcinogenesis.

The risk of breast cancer transiently increases immediately following pregnancy; peaking between 3-7 years. The biology that underlies this risk window and the effect on the natural history of the disease is unknown. MicroRNAs (miRNAs) are small, non-coding RNAs that have been shown to be dysregulated in breast cancer. Utilizing a quantitative RT-PCR approach, we profiled the expression pattern of 355 miRNAs across 56 formalin-fixed, paraffin embedded breast tumor tissue samples from multiparous Hispanic women. Based on a data-driven splitting of the expression pattern of the 355 miRNAs, the case series was separated into two groups based on time since last full-term pregnancy (TSLFTP): a) an early group representing women diagnosed with breast cancer ≤ 5.2 years postpartum (n=12); and b) a late group representing women diagnosed with breast cancer ≥ 5.3 years postpartum (n=44). We identified 15 miRNAs with significant differential expression between the early and late postpartum groups. Ten miRNAs had a two-fold or higher difference in expression, with miR-138, miR-660, miR-31, miR-135b, miR-17, miR-454, and miR-934 overexpressed in the early versus the late group; while miR-892a, miR-199a-5p, and miR-542-5p were overexpressed in the late versus the early postpartum group. Further analysis of the miRNA expression data demonstrated that 60% of the identified differentially expressed miRNAs are encoded on the X chromosome, with several miRNAs found together in miRNA gene clusters. Additionally, DNA methylation, measured using a Sequenom MassARRAY, demonstrated that three out of five tested miRNA genes (mir-31, mir-135b, and mir-138-2) had a lower mean methylation in the early versus late postpartum group, and negatively correlated with miRNA expression. In summary, we have identified miRNAs that are differentially expressed and differentially methylated between tumors of the early versus late postpartum, suggesting that potential differences in epigenetic regulation may exist in the postpartum breast cancers. Citation Format: José L. Muñoz-Rodríguez, Lukas Vrba, Chengcheng Hu, Patricia A. Thompson, Maria Elena Martinez, Bernard W. Futscher. Identifying differentially expressed microRNAs as investigational tools in postpartum breast cancer in Hispanic women. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A82.

The risk of breast cancer transiently increases immediately following pregnancy; peaking between 3-7 years. The biology that underlies this risk window and the effect on the natural history of the disease is unknown. MicroRNAs (miRNAs) are small non-coding RNAs that have been shown to be dysregulated in breast cancer. We conducted miRNA profiling of 56 tumors from a case series of multiparous Hispanic women and assessed the pattern of expression by time since last full-term pregnancy. A data-driven splitting analysis on the pattern of 355 miRNAs separated the case series into two groups: a) an early group representing women diagnosed with breast cancer ≤ 5.2 years postpartum (n = 12), and b) a late group representing women diagnosed with breast cancer ≥ 5.3 years postpartum (n = 44). We identified 15 miRNAs with significant differential expression between the early and late postpartum groups; 60% of these miRNAs are encoded on the X chromosome. Ten miRNAs had a two-fold or higher difference in expression with miR-138, miR-660, miR-31, miR-135b, miR-17, miR-454, and miR-934 overexpressed in the early versus the late group; while miR-892a, miR-199a-5p, and miR-542-5p were underexpressed in the early versus the late postpartum group. The DNA methylation of three out of five tested miRNAs (miR-31, miR-135b, and miR-138) was lower in the early versus late postpartum group, and negatively correlated with miRNA expression. Here we show that miRNAs are differentially expressed and differentially methylated between tumors of the early versus late postpartum, suggesting that potential differences in epigenetic dysfunction may be operative in postpartum breast cancers.

The risk of breast cancer has been shown to increase immediately following pregnancy. Breast cancers that occur in the post-partum period are more aggressive, more likely to metastasize and are associated with worse patient outcomes. They also tend to affect younger women, and may represent a substantial proportion of early onset breast cancer, a phenomenon that disproportionately affects racial and ethnic minority groups, such as Hispanics. Hispanic women are more likely to have higher parity, are diagnosed with breast cancer at a younger age, and have a worse survival after diagnosis. The goal of this study, therefore, is to compare breast cancers diagnosed in Hispanic women in the high-risk post-partum period with those diagnosed outside of this time period to identify epigenetic features associated with the aggressive post-partum cancer. RNA and DNA were isolated from formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues from a Hispanic population within the Ella Binational Breast Cancer study participants. Using a quantitative real-time reverse transcriptase approach (Exiqon miRCURY LNA™ Universal RT microRNA PCR panels), we were able to test for the expression of 360 human microRNAs (miRNAs) that our laboratory previously identified as miRNAs that are specific for human mammary cells. MiRNAs are short-stranded, non-coding RNAs that are often deregulated in breast cancer. We have identified seven miRNAs that are differentially expressed in breast cancer that occurs in the post-partum period including: hsa-miR-137, hsa-miR-31, hsa-miR-342-3p, hsa-miR-342-5p, hsa-miR-542-5p, hsa-miR-190b, and hsa-miR-103. Previously, our laboratory showed that epigenetic modifications, such as DNA methylation, deregulate miRNA expression in breast cancer. Consequently, we used a Sequenom MassARRAY platform to determine whether these miRNAs are also being epigenetically regulated. We are also investigating the mechanistic role these miRNAs may play in the early onset post-partum breast cancers. Citation Format: Jose L. Munoz-Rodriguez, Lukas Vrba, Chengcheng Hu, Patricia A. Thompson-Carino, Maria Elena Martinez, Bernard W. Futscher. Epigenetic changes in postpartum breast cancer amongst Hispanic women. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C74. doi:10.1158/1538-7755.DISP13-C74

Dysregulation of epigenetic control is an important participant in carcinogenesis. The PML/RAR alpha translocation in acute promyelocytic leukemia (APL) is an example where the resultant fusion protein recruits histone deacetylase complexes to target genes resulting in their inappropriate transcriptional repression. All-trans-retinoic acid (ATRA) acts as a ligand that relieves this repression and produces an epigenetic transcriptional reprogramming of the cancer cell. CpG island microarrays were used to analyze the DNA methylation and histone acetylation state of the human APL cell line NB4 before and after differentiation with ATRA as well as normal peripheral blood mononuclear cells (PBMC). Over 70 CpG islands within 1 kb of transcription start of a known gene are aberrantly methylated in NB4 cells compared with PBMC; however, no changes in cytosine methylation were detected following ATRA-induced differentiation. With respect to histone H4 acetylation, over 100 single-copy CpG islands within 1 kb of transcription start of a known human gene became hyperacetylated following ATRA-induced differentiation. One CpG island was aberrantly methylated in NB4 cells, but became hyperacetylated and was induced following ATRA treatment and was associated with the HoxA1 gene, suggesting it may be a target gene of ATRA in APL. In addition to single-copy sequences, a selective increase in acetylation was detected in satellite DNA when compared with other high-copy sequences, such as Alu or rDNA. In summary, ATRA stimulates complex epigenomic changes during leukemic cell differentiation, and monitoring these changes may help to identify new targets of epigenetic dysfunction.

p53 is an important transcriptional regulator that is frequently mutated in cancer. Gene-profiling experiments of breast cancer cells infected with wt p53 revealed both MASPIN and desmocollin 3 (DSC3) to be p53-target genes, even though both genes are silenced in association with aberrant cytosine methylation of their promoters. Despite the transcriptional repression of these genes by aberrant DNA methylation, restoration of p53 resulted in the partial reactivation of both genes. This reactivation is a result of wt p53 binding to its consensus DNA-binding sites within the MASPIN and DSC3 promoters, stimulating histone acetylation, and enhancing chromatin accessibility of their promoters. Interestingly, wt p53 alone did not affect the methylation status of either promoter, suggesting that p53 itself can partially overcome the repressive barrier of DNA methylation. Pharmacologic inhibition of DNA methylation with 5-aza-2'-deoxycytidine in combination with restoration of wt p53 status resulted in a synergistic reactivation of these genes to near-normal levels. These results suggest that cancer treatments that target both genetic and epigenetic facets of gene regulation may be a useful strategy towards the therapeutic transcriptional reprogramming of cancer cells.

miRNAs are important regulators of gene expression that are frequently deregulated in cancer, with aberrant DNA methylation being an epigenetic mechanism involved in this process. We previously identified miRNA promoter regions active in normal mammary cell types and here we analyzed which of these promoters are targets of aberrant DNA methylation in human breast cancer cell lines and breast tumor specimens. Using 5-methylcytosine immunoprecipitation coupled to miRNA tiling microarray hybridization, we performed comprehensive evaluation of DNA methylation of miRNA gene promoters in breast cancer. We found almost one third (55/167) of miRNA promoters were targets for aberrant methylation in breast cancer cell lines. Breast tumor specimens displayed DNA methylation of majority of these miRNA promoters, indicating that these changes in DNA methylation might be clinically relevant. Aberrantly methylated miRNA promoters were, similar to protein coding genes, enriched for promoters targeted by polycomb in normal cells. Detailed analysis of selected miRNA promoters revealed decreased expression of miRNA linked to increased promoter methylation for mir-31, mir-130a, let-7a-3/let-7b, mir-155, mir-137 and mir-34b/mir-34c genes. The proportion of miRNA promoters we found aberrantly methylated in breast cancer is several fold larger than that observed for protein coding genes, indicating an important role of DNA methylation in miRNA deregulation in cancer.

MicroRNAs (miRNAs) are short-stranded, non-coding RNAs that regulate gene expression. The expression of miRNAs is often deregulated in breast cancer. Epigenetic modifications, such as DNA methylation, have been shown to regulate miRNA gene expression in breast cancer. In our study, we set out to analyze the role that differentially expressed miRNAs play in pregnancy associated breast cancer (PABC) amongst a Hispanic population within the Ella Binational Breast Cancer study. PABC is defined as cancer that arises within a time-frame of 10 years after pregnancy. Previous studies have shown that women diagnosed with PABC do far worse than women diagnosed with non-PABC. Our hypothesis is that the epigenetic regulation of miRNA genes contributes to poor survival in PABC. We are testing our hypothesis using a cohort of post-partum formalin fixed, paraffin embedded (FFPE) breast cancer tissues from a Hispanic population. Using Exiqon miRCURY LNA™ Universal RT microRNA PCR panels we were able to test for the expression of 360 human miRNAs, which our laboratory previously identified as miRNAs that are specific for human mammary cells. Preliminary data from a small subset of samples showed six miRNAs that were differentially expressed (having > 4-fold expression and a p-value ≤ 0.05) between PABC and non-PABC (hsa-miR-876-5p; hsa-miR-299-3p; hsa-miR-137; hsa-miR-187; hsa-miR-873; hsa-miR-190b ). Several of these miRNAs have been implicated in carcinogenesis. Our goals are to identify differentially expressed miRNAs and to determine the extent of the epigenetic regulation of those miRNAs. These miRNAs will be evaluated for their potential utility as targets of epigenetic regulation and targets for therapeutic interventions. In addition, our studies will be the first to look at epigenetic mechanisms related to PABC in Hispanics. Hispanics have one of the highest fertility rates and an early breast cancer diagnosis. Therefore, our results will elucidate factors associated with this PABC disparity. Citation Format: Jose Luis Munoz-Rodriguez, Lukas Vrba, Patricia A. Thompson-Carino, Maria Elena Martinez, Bernard W. Futscher. Pregnancy-associated breast cancer and differentially expressed microRNAs. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B34.

MicroRNAs (miRNA) are short single-stranded RNA molecules that regulate gene expression and deregulation of their expression is linked to human diseases including cancer. Epigenetic mechanisms including DNA methylation play an important role in the regulation of miRNA expression, and a number of reports describe silencing of miRNA expression linked to the aberrant DNA methylation of individual miRNA genes in cancer. In this study, we have used a comprehensive miRNA custom tiling microarray to evaluate aberrant DNA methylation of miRNA gene promoters in human breast cancer, with selected hypermethylated promoters being validated by Sequenom MassARRAY technology. We found more than 25% of miRNA gene promoters analyzed have significant hypermethylated regions in breast cancer cell lines (CCL) relative to logarithmically growing normal pre-stasis finite lifespan human mammary epithelial cells isolated from healthy individuals. In addition to in vitro grown breast cancer cell lines, we analyzed DNA methylation of miRNA promoters in a dozen breast tumor tissue specimens and five normal breast specimens. Aberrant DNA hypermethylation of miRNA promoters in breast cancer specimens displayed a perfect overlap with those hypermethylated in breast cancer cell lines, albeit the number of miRNAs promoters was smaller in the heterogeneous tissue samples. These aberrantly methylated miRNA promoters readily clustered breast cancer samples and cell lines together, and separate from normal beast tissue and mammary cell strains. Integration of our data with publically-available H3K27me3 ChIP-Seq data from stem cells reveals that miRNA promoters targeted by aberrant DNA methylation in cancer cells significantly overlap with promoters targeted by polycomb repression complexes in stem cells, similar to the results other have seen with protein coding genes promoters in cancer. We have found previously that the proportion of miRNA genes targeted by polycomb repression in normal mammary cell types is about 27% - three times higher than seen in protein coding genes, perhaps explaining why the proportion of miRNA genes with aberrant DNA methylation in our analysis of breast cancer is so high. In summary, our data show that a large fraction of miRNA gene promoters are targets of aberrant DNA methylation in human breast cancer, and that the miRNA promoters aberrantly methylated in breast cancer cell lines are also targeted in tumor tissue samples, indicating that these changes in DNA methylation are not artifacts of cell culture and might thus have clinical relevance. Similar to protein coding genes, miRNA gene promoters targeted by polycomb complexes in stem cells are more prone to hypermethylation in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 123. doi:1538-7445.AM2012-123