Rohit R. Jadhav

Single-cell analysis of circulating tumor cells identifies cumulative expression patterns of EMT-related genes in metastatic prostate cancer

Prostate tumors shed circulating tumor cells (CTCs) into the blood stream. Increased evidence shows that CTCs are often present in metastatic prostate cancer and can be alternative sources for disease profiling and prognostication. Here we postulate that CTCs expressing genes related to epithelial–mesenchymal transition (EMT) are strong predictors of metastatic prostate cancer.

Promoter hypomethylation of EpCAM-regulated bone morphogenetic protein gene family in recurrent endometrial cancer

Purpose: Epigenetic regulation by promoter methylation plays a key role in tumorigenesis. Our goal was to investigate whether altered DNA methylation signatures associated with oncogenic signaling delineate biomarkers predictive of endometrial cancer recurrence. Experimental Design: Methyl-CpG-capture sequencing was used for global screening of aberrant DNA methylation in our endometrial cancer cohort, followed by validation in an independent The Cancer Genome Atlas (TCGA) cohort. Bioinformatics as well as functional analyses in vitro, using RNA interference (RNAi) knockdown, were performed to examine regulatory mechanisms of candidate gene expression and contribution to aggressive phenotype, such as epithelial–mesenchymal transition (EMT). Results: We identified 2,302 hypermethylated loci in endometrial tumors compared with control samples. Bone morphogenetic protein (BMP) family genes, including BMP1, 2, 3, 4, and 7, were among the frequently hypermethylated loci. Interestingly, BMP2, 3, 4, and 7 were less methylated in primary tumors with subsequent recurrence and in patients with shorter disease-free interval compared with nonrecurrent tumors, which was validated and associated with poor survival in the TCGA cohort (BMP4, P = 0.009; BMP7, P = 0.007). Stimulation of endometrial cancer cells with epidermal growth factor (EGF) induced EMT and transcriptional activation of these genes, which was mediated by the epithelial cell adhesion molecule (EpCAM). EGF signaling was implicated in maintaining the promoters of candidate BMP genes in an active chromatin configuration and thus subject to transcriptional activation. Conclusions: Hypomethylation signatures of candidate BMP genes associated with EpCAM-mediated expression present putative biomarkers predictive of poor survival in endometrial cancer. Clin Cancer Res; 19(22); 6272–85. ©2013 AACR.

Epigenetic deregulation of the anaplastic lymphoma kinase gene modulates mesenchymal characteristics of oral squamous cell carcinomas

DNA hypermethylation of promoter CpG islands is associated with epigenetic silencing of tumor suppressor genes in oral squamous cell carcinomas (OSCCs). We used a methyl-CpG-binding domain protein capture method coupled with next-generation sequencing (MBDCap-seq) to survey global DNA methylation patterns in OSCCs with and without nodal metastasis and normal mucosa (total n = 58). Of 1462 differentially methylated CpG islands identified in OSCCs relative to normal controls, MBDCap-seq profiling uncovered 359 loci linked to lymph node metastasis. Interactive network analysis revealed a subset of these loci (n = 23), including the anaplastic lymphoma kinase (ALK) gene, are potential regulators and effectors of invasiveness and metastatic progression. Promoter methylation of ALK was preferentially observed in OSCCs without node metastasis, whereas relatively lower methylation levels were present in metastatic tumors, implicating an active state of ALK transcription in the latter group. The OSCC cell line, SCC4, displayed reduced ALK expression that corresponded to extensive promoter CpG island methylation. SCC4 treatment with demethylating agents induced ALK expression and increased invasion and migration characteristics. Inhibition of ALK activity in OSCC cells with high ALK expression (CAL27, HSC3 and SCC25), decreased cell growth and resulted in changes in invasive potential and mesenchymal marker expression that were cell-line dependent. Although ALK is susceptible to epigenetic silencing during oral tumorigenesis, overwriting this default state may be necessary for modulating invasive processes involved in nodal metastases. Given the complex response of OSCC cells to ALK inhibition, future studies are required to assess the feasibility of targeting ALK to treat invasive OSCCs.

Roles of distal and genic methylation in the development of prostate tumorigenesis revealed by genome-wide DNA methylation analysis

Aberrant DNA methylation at promoters is often linked to tumorigenesis. But many aspects of DNA methylation remain unexplored, including the individual roles of distal and gene body methylation, as well as their collaborative roles with promoter methylation. Here we performed a MBD-seq analysis on prostate specimens classified into low, high, and very high risk group based on Gleason score and TNM stages. We identified gene sets with differential methylation regions (DMRs) in Distal, TSS, gene body and TES. To understand the collaborative roles, TSS was compared with the other three DMRs, resulted in 12 groups of genes with collaborative differential methylation patterns (CDMPs). We found several groups of genes that show opposite methylation patterns in Distal and Genic regions compared to TSS region, and in general they are differentially expressed genes (DEGs) in tumors in TCGA RNA-seq data. IPA (Ingenuity Pathway Analysis) reveals AR/TP53 signaling network to be a major signaling pathway, and survival analysis indicates genes subsets significantly associated with prostate cancer recurrence. Our results suggest that DNA methylation in Distal and Genic regions also plays critical roles in contributing to prostate tumorigenesis, and may act either positively or negatively with TSSs to alter gene regulation in tumors.

DNA methylation targets influenced by bisphenol A and/or genistein are associated with survival outcomes in breast cancer patients

Early postnatal exposures to Bisphenol A (BPA) and genistein (GEN) have been reported to predispose for and against mammary cancer, respectively, in adult rats. Since the changes in cancer susceptibility occurs in the absence of the original chemical exposure, we have investigated the potential of epigenetics to account for these changes. DNA methylation studies reveal that prepubertal BPA exposure alters signaling pathways that contribute to carcinogenesis. Prepubertal exposure to GEN and BPA + GEN revealed pathways involved in maintenance of cellular function, indicating that the presence of GEN either reduces or counters some of the alterations caused by the carcinogenic properties of BPA. We subsequently evaluated the potential of epigenetic changes in the rat mammary tissues to predict survival in breast cancer patients via the Cancer Genomic Atlas (TCGA). We identified 12 genes that showed strong predictive values for long-term survival in estrogen receptor positive patients. Importantly, two genes associated with improved long term survival, HPSE and RPS9, were identified to be hypomethylated in mammary glands of rats exposed prepuberally to GEN or to GEN + BPA respectively, reinforcing the suggested cancer suppressive properties of GEN.

Immune Checkpoint Function of CD85j in CD8 T Cell Differentiation and Aging

Aging is associated with an increased susceptibility to infection and a failure to control latent viruses thought to be driven, at least in part, by alterations in CD8 T cell function. The aging T cell repertoire is characterized by an accumulation of effector CD8 T cells, many of which express the negative regulatory receptor CD85j. To define the biological significance of CD85j expression on CD8 T cells and to address the question whether presence of CD85j in older individuals is beneficial or detrimental for immune function, we examined the specific attributes of CD8 T cells expressing CD85j as well as the functional role of CD85j in antigen-specific CD8 T cell responses during immune aging. Here, we show that CD85j is mainly expressed by terminally differentiated effector (TEMRAs) CD8 T cells, which increase with age, in cytomegalovirus (CMV) infection and in males. CD85j+ CMV-specific cells demonstrate clonal expansion. However, TCR diversity is similar between CD85j+ and CD85j− compartments, suggesting that CD85j does not directly impact the repertoire of antigen-specific cells. Further phenotypic and functional analyses revealed that CD85j identifies a specific subset of CMV-responsive CD8 T cells that coexpress a marker of senescence (CD57) but retain polyfunctional cytokine production and expression of cytotoxic mediators. Blocking CD85j binding enhanced proliferation of CMV-specific CD8 T cells upon antigen stimulation but did not alter polyfunctional cytokine production. Taken together, these data demonstrate that CD85j characterizes a population of “senescent,” but not exhausted antigen-specific effector CD8 T cells and indicates that CD85j is an important checkpoint regulator controlling expansion of virus-specific T cells during aging. Inhibition of CD85j activity may be a mechanism to promote stronger CD8 T cell effector responses during immune aging.

Single-cell RNA-seq reveals a subpopulation of prostate cancer cells with enhanced cell cycle-related transcription and attenuated androgen response

Increasing evidence suggests the presence of minor cell subpopulations in prostate cancer that are androgen independent and poised for selection as dominant clones after androgen deprivation therapy. In this study, we investigated this phenomenon by stratifying cell subpopulations based on transcriptome profiling of 144 single LNCaP prostate cancer cells treated or untreated with androgen after cell-cycle synchronization. Model-based clustering of 397 differentially expressed genes identified eight potential subpopulations of LNCaP cells, revealing a previously unappreciable level of cellular heterogeneity to androgen stimulation. One subpopulation displayed stem-like features with a slower cell doubling rate, increased sphere formation capability, and resistance to G2-M arrest induced by a mitosis inhibitor. Advanced growth of this subpopulation was associated with enhanced expression of 10 cell-cycle-related genes (CCNB2, DLGAP5, CENPF, CENPE, MKI67, PTTG1, CDC20, PLK1, HMMR, and CCNB1) and decreased dependence upon androgen receptor signaling. In silico analysis of RNA-seq data from The Cancer Genome Atlas further demonstrated that concordant upregulation of these genes was linked to recurrent prostate cancers. Analysis of receiver operating characteristic curves implicates aberrant expression of these genes and could be useful for early identification of tumors that subsequently develop biochemical recurrence. Moreover, this single-cell approach provides a better understanding of how prostate cancer cells respond heterogeneously to androgen deprivation therapies and reveals characteristics of subpopulations resistant to this treatment.Significance: Illustrating the challenge in treating cancers with targeted drugs, which by selecting for drug resistance can drive metastatic progression, this study characterized the plasticity and heterogeneity of prostate cancer cells with regard to androgen dependence, defining the character or minor subpopulations of androgen-independent cells that are poised for clonal selection after androgen-deprivation therapy. Cancer Res; 78(4); 853-64. ©2017 AACR.

Regulation of miR-181a expression in T cell aging

MicroRNAs have emerged as key regulators in T cell development, activation, and differentiation, with miR-181a having a prominent function. By targeting several signaling pathways, miR-181a is an important rheostat controlling T cell receptor (TCR) activation thresholds in thymic selection as well as peripheral T cell responses. A decline in miR-181a expression, due to reduced transcription of pri-miR-181a, accounts for T cell activation defects that occur with older age. Here we examine the transcriptional regulation of miR-181a expression and find a putative pri-miR-181a enhancer around position 198,904,300 on chromosome 1, which is regulated by a transcription factor complex including YY1. The decline in miR-181a expression correlates with reduced transcription of YY1 in older individuals. Partial silencing of YY1 in T cells from young individuals reproduces the signaling defects seen in older T cells. In conclusion, YY1 controls TCR signaling by upregulating miR-181a and dampening negative feedback loops mediated by miR-181a targets.MicroRNA miR-181a has been implicated in the regulation of T cell activation and development. Here the authors show that miR-181a is regulated by a transcription factor, YY1, with reduced YY1 expression linked to reduced miR-181a in old individuals, while silencing YY1 impairs T cell functions largely through influencing the expression of miR-181a targets.

Epigenetics of T cell aging

T cells are a heterogeneous population of cells that differ in their differentiation stages. Functional states are reflected in the epigenome that confers stability in cellular identity and is therefore important for naïve as well as memory T cell function. In many cellular systems, changes in chromatin structure due to alterations in histone expression, histone modifications and DNA methylation are characteristic of the aging process and cause or at least contribute to cellular dysfunction in senescence. Here, we review the epigenetic changes in T cells that occur with age and discuss them in the context of canonical epigenetic marks in aging model systems as well as recent findings of chromatin accessibility changes in T cell differentiation. Remarkably, transcription factor networks driving T cell differentiation account for many of the age‐associated modifications in chromatin structures suggesting that loss of quiescence and activation of differentiation pathways are major components of T cell aging.

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).