Single-cell dissection of cellular components and interactions shaping the tumor immune phenotypes in ovarian cancer.

Distinct T cell infiltration patterns, i.e., immune infiltrated, excluded, and desert, result in different responses to cancer immunotherapies. However, the key determinants and biology underpinning these tumor immune phenotypes remain elusive. Here, we provide a high-resolution dissection of the entire tumor ecosystem through single-cell RNA-sequencing analysis of 15 ovarian tumors. Immune-desert tumors are characterized by unique tumor cell-intrinsic features, including metabolic pathways and low antigen presentation, and an enrichment of monocytes and immature macrophages. Immune-infiltrated and -excluded tumors differ markedly in their T cell composition and fibroblast subsets. Furthermore, our study reveals chemokine receptor-ligand interactions within and across compartments as potential mechanisms mediating immune cell infiltration, exemplified by the tumor cell-T cell cross talk via CXCL16-CXCR6 and stromal-immune cell cross talk via CXCL12/14-CXCR4. Our data highlight potential molecular mechanisms that shape the tumor immune phenotypes and may inform therapeutic strategies to improve clinical benefit from cancer immunotherapies.

Mutant p53 and aberrant cytosine methylation cooperate to silence gene expression.

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.

Epigenetic silencing of DSC3 is a common event in human breast cancer.

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.

Differentially expressed microRNAs in postpartum breast cancer in Hispanic women.

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.

miRNA gene promoters are frequent targets of aberrant DNA methylation in human breast cancer.

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.

Thiostrepton is an inducer of oxidative and proteotoxic stress that impairs viability of human melanoma cells but not primary melanocytes.

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.

Cell-type specific DNA methylation patterns define human breast cellular identity.

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.