Sridhar Ramaswamy

• Massachusetts General Hospital

[This corrects the article DOI: 10.1371/journal.pone.0140310.].

Ductal carcinoma in situ (DCIS) of the breast precedes the development of invasive breast cancer and reflects the genomic changes and protein expression profile of invasive disease. AKT1low cancer cells (QCC) are a rare, drug-tolerant, epigenetically plastic, and quiescent cancer cell subset that we previously identified at a frequency of 0.5–1% in...

Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release...

Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single-cell RNA sequencing in an orthotopic mouse prostate cancer model, we find up-regulation of prolactin receptor as cancer cells that have disseminated to the lungs expand into micrometastases....

Background Drug combinations have the potential to improve efficacy while limiting toxicity. To robustly identify synergistic combinations, high-throughput screens using full dose-response surface are desirable but require an impractical number of data points. Screening of a sparse number of doses per drug allows to screen large numbers of drug pai...

A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-L1 expression and mutational burden, are insufficiently precise to guide treatment selection and clinical radiographic eviden...

Significance Identifying predictive biomarkers of therapeutic response for melanoma patients treated with immune checkpoint inhibitors is a major challenge. By combining microfluidic enrichment for melanoma circulating tumor cells (CTCs) together with RNA-based droplet digital PCR quantitation, we have established a highly sensitive and robust plat...

Molecular drivers underlying bone metastases in human cancer are not well understood, in part due to constraints in bone tissue sampling. Here, RNA sequencing was performed of circulating tumor cells (CTC) isolated from blood samples of women with metastatic estrogen receptor (ER)+ breast cancer, comparing cases with progression in bone versus visc...

To understand the consequences of the complete elimination of E2F regulation, we profiled the proteome of Drosophila dDP mutants that lack functional E2F/DP complexes. The results uncovered changes in the larval fat body, a differentiated tissue that grows via endocycles. We report an unexpected mechanism of E2F/DP action that promotes quiescence i...

Human tumor growth depends on rapidly dividing cancer cells undergoing population expansion. Even advanced tumors, however, contain slowly proliferating cancer cells for reasons that remain unclear. Here, we selectively disrupt the ability of rapidly proliferating cancer cells to spawn AKT1low daughter cells that are rare, slowly proliferating, tum...

Epithelial to mesenchymal transition (EMT), a morphogenetic process required for proper embryonic development, is adopted by cancer cells during tumor progression. We show that induction of EMT by TGFß or other EMT-inducers such as Snail leads to genomic instability, associated with failed cytokinesis and chromosome missegregation resulting in aneu...

11579 Background: The mechanisms that allow triple negative breast cancer (TNBC) tumors to survive neoadjuvant chemotherapy (NACT) are incompletely understood. Evidence suggests that proliferative heterogeneity may contribute to primary chemotherapy resistance in patients with localized triple negative breast cancer. However, the detailed character...

TGF-β is a cytokine thought to function as a tumor promoter in advanced malignancies. In this setting, TGF-β increases cancer cell proliferation, survival, and migration, and orchestrates complex, pro-tumorigenic changes in the tumor microenvironment. Here, we find that in melanoma, integrin β1-mediated TGF-β activation may also produce tumor suppr...

TS2/16 specifically binds human β1 integrin. (A) B16F0 and A375 tumor cells were treated with IgG or TS2/16, then fixed and stained for IgG1 alexa fluor 488 (AF 488), a secondary antibody recognizing TS2/16. Representative picture from n = 3 experiments. (B) Histogram from FACS experiment of B16F0 (upper graph) and A375 (lower graph) tumor cells tr...

TS2/16 activates integrin β1. (A) TGF-β co-culture assay. Quantification of the relative luciferase units (RLU—measure for active TGF-β) of tMLEC/A375 co-cultures (open bars) or tMLEC/SK-Mel-28 co-cultures treated with IgG or TS2/16. Graphs are normalized to IgG treatment. N = 3 experiments performed in triplicate. (B) Quantification of FACS data a...

List of used TCGA melanoma samples. List of TCGA melanoma samples and clinical information for samples that have both RNASeqV2 RSEM expression data and clinical data. (XLSX)

TGF-β1 expression does not correlate with tumor stage. (A) GSEA analyses for TCGA SKCM patient samples (excluding lymph node metastases) with genes ranked based on Pearson correlation with TGFB1 expression level and testing enrichment of the tumor microenvironment gene signatures (S2 Table). (B) Heatmap showing TCGA SKCM patient sample correlations...

Supplementary methods. This file contains supplementary methods regarding the bioinformatics analyses that were performed. (DOCX)

Integrin β1 activation by TS2/16 results in typical TGF-β-associated microenvironmental changes. (A) IHC of tumors left untreated or treated with TS2/16 for 5 weeks. The number of CD31+ microvessels or aSMA+CD31- CAFs per field of view (FOV) was quantified. For Type I collagen the mean fluorescence intensity per FOV for COL1A1 was calculated. N = 1...

TGFB1 associations and survival analyses. (A) Histogram displaying the distribution of TGFB1 expression level of TCGA SKCM samples subdivided into groups based on tumor or metastasis type. (B) Kaplan-Meier curve displaying OS for all TCGA SKCM patients who were subdivided into TGFB1high and TGFB1low groups. TGFB1high > median (N = 222), TGFB1low <...

TS2/16 does not affect CD8+ cell proliferation or apoptosis. (A) Micrographs of IgG or TS2/16 treated tumors stained for DAPI, CD8 and CD3. CD3+CD8+ T cells are indicated by a yellow arrow. A zoom in of the boxed area is shown on the right. Scale bar, 50 μm. (B) Quantification of FACS data averaging the mean fluorescence intensity for A375 and SK-M...

Gene signatures. List of genes in the tumor microenvironment signature and their correlations with TGFB1 expression. (XLSX)

TCGA SKCM TGFB1 clinical associations. Association of clinical and pathological parameters with TGFB1 RNA-seq expression (t-test or Pearson correlation test). (XLSX)

Summary table of all TCGA cancer type data sets. Summary table of outcome, GSEA results, ITGB1 meta-gene correlation and SERPINE1 correlation in all TCGA cancer type data sets. (XLSX)

Poly-(ADP-ribose) polymerase (PARP) inhibitors (PARPis) selectively kill BRCA1/2-deficient cells, but their efficacy in BRCA-deficient patients is limited by drug resistance. Here, we used derived cell lines and cells from patients to investigate how to overcome PARPi resistance. We found that the functions of BRCA1 in homologous recombination (HR)...

Metastasis-competent circulating tumour cells (CTCs) experience oxidative stress in the bloodstream, but their survival mechanisms are not well defined. Here, comparing single-cell RNA-Seq profiles of CTCs from breast, prostate and lung cancers, we observe consistent induction of β-globin (HBB), but not its partner α-globin (HBA). The tumour-specif...

Supplementary Figures and Supplementary Tables

Chromatin remodeling proteins are frequently dysregulated in human cancer; however, little is known about how they control tumorigenesis. The NAD+ dependent histone deacetylase Sirtuin 6 (SIRT6) is a nutrient sensor that reprograms the epigenome in response to nutrient stress and shows reduced expression in human pancreatic ductal adenocarcinoma (P...

Chromatin remodeling proteins are frequently dysregulated in human cancer; however, little is known about how they control tumorigenesis. The NAD+ dependent histone deacetylase Sirtuin 6 (SIRT6) is a nutrient sensor that reprograms the epigenome in response to nutrient stress and shows reduced expression in human pancreatic ductal adenocarcinoma (P...

TGF-β secreted by tumor stroma induces epithelial-to-mesenchymal transition (EMT) in cancer cells, a reversible phenotype linked to cancer progression and drug resistance. However, exposure to stromal signals may also lead to heritable changes in cancer cells, which are poorly understood. We show that epithelial cells failing to undergo proliferati...

Fibroblast-like mesenchymal cells are the most abundant component of tumor stroma and have been shown to promote tumor progression. However, there are no therapeutic agents that target these cells. Developing therapeutics against mesenchymal cells has been challenging because they lack clear “druggable” targets. Here we used a phenotypic screening...

Chromatin remodeling proteins are frequently dysregulated in human cancer, however little is known about how they control tumorigenesis. The NAD+-dependent histone deacetylase Sirtuin 6 (SIRT6) is a nutrient sensor that reprograms the epigenome in response to nutrient stress and shows reduced expression in human pancreatic ductal adenocarcinoma (PD...

Identification of candidate metastasis genes has traditionally resulted from comparison of primary and metastatic tumor specimens. However, Circulating Tumor Cells (CTCs) contain metastatic precursors that are present transiently in the bloodstream and their analysis may reveal additional pathways that are induced for a limited time, as they invade...

Chromatin remodeling proteins are frequently dysregulated in human cancer, yet little is known about how they control tumorigenesis. Here, we uncover an epigenetic program mediated by the NAD+-dependent histone deacetylase Sirtuin 6 (SIRT6) that is critical for suppression of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal malignanc...

Isolation of circulating tumor cells (CTCs) in the blood allows noninvasive tumor sampling from patients with cancer. Molecular analysis of single CTCs may uncover heterogeneous cellular pathways that underlie disease progression and resistance to therapy. Using a microfluidic device, we isolated individual CTCs from patients with metastatic prosta...

Small-molecule inhibitors of AKT signaling are being evaluated in patients with various cancer types, but have so far proven therapeutically disappointing for reasons that remain unclear. Here, we treat cancer cells with sub-therapeutic doses of Akti-1/2, an allosteric small molecule AKT inhibitor, in order to experimentally model pharmacologic inh...

Significance Unique among the large number of noncoding RNA species, the pericentromeric human satellite II (HSATII) repeat is massively expressed in a broad set of epithelial cancers but is nearly undetectable in normal tissues. Here, we show that deregulation of HSATII expression is tightly linked to growth under nonadherent conditions, and we un...

Large cancer cell line collections broadly capture the genomic diversity of human cancers and provide valuable insight into anti-cancer drug response. Here we show substantial agreement and biological consilience between drug sensitivity measurements and their associated genomic predictors from two publicly available large-scale pharmacogenomics re...

A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year. Thus, there is a pressing need for identification of combinations...

Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targ...

Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging; hence, sampling circulating tumor cells (CTCs) may reveal drug-resistance mechanisms. We established single-cell RNA-sequencing (RNA-Seq) profiles of...

Disease relapse in cancer patients many years after clinical remission, often referred to as cancer dormancy, is well documented but remains an incompletely understood phenomenon on the biologic level. Recent reviews have summarized potential models that can explain this phenomenon, including angiogenic, immunologic, and cellular dormancy. We focus...

How do cancer cells escape tightly controlled regulatory circuits that link their growth to extracellular signaling cues? An emerging theme in cancer biology is how, in addition to genetic alterations in signaling pathways (eg. MAPK and PI3K), cancer cells can hijack normal stress response pathways to overcome reliance on external nutrients for gro...

Activation of cellular stress response pathways to maintain metabolic homeostasis is emerging as a critical growth and survival mechanism in many cancers. The pathogenesis of pancreatic ductal adenocarcinoma (PDA) requires high levels of autophagy, a conserved self-degradative process. However, the regulatory circuits that activate autophagy and re...

Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC clusters). Existing technologies for CTC enrichment are designed to isolate single CTCs, and although CTC clusters are detectable in some cases, their true prevalence and significance remain to be determined....

WTX encodes a tumor suppressor implicated in the pediatric kidney cancer Wilms tumor and in mesenchymal differentiation, with potentially distinct functions in the cytoplasm, at the plasma membrane and in the nucleus. While modulating components of the WNT signaling pathway is a proposed function for cytoplasmic and membrane-bound WTX, its nuclear...

Epithelial-to-Mesenchymal Transition (EMT) has been implicated in models of tumor cell migration, invasion and metastasis. In a search for candidate therapeutic targets to reverse this process, non-tumorigenic MCF10A breast epithelial cells were infected with an arrayed lentiviral kinome shRNA library and screened for either suppression or enhancem...

All cancers contain an admixture of rapidly and slowly proliferating cancer cells. This proliferative heterogeneity complicates the diagnosis and treatment of patients with cancer because slow proliferators are hard to eradicate, can be difficult to detect, and may cause disease relapse sometimes years after apparently curative treatment. While clo...

Modeling the hematogenous spread of cancer cells to distant organs poses one of the greatest challenges in the study of human metastasis. Both tumor-cell intrinsic properties as well as interactions with reactive stromal cells contribute to this process, but identification of relevant stromal signals has been hampered by the lack of models allowing...

The metastatic spread of breast cancer, typically to bone, lung, liver and brain, accounts for the vast majority of cancer-related deaths. Breast cancer metastases are thought to be derived primarily from individual migratory cancer cells, reaching distant sites through the bloodstream and initiating proliferation within distant organs. In addition...

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal adult malignancy due to the propensity of this disease to metastasize. Circulating tumor cells (CTCs) are thought to be enriched for cells with metastatic potential and their characterization offers a means to understand the biological underpinnings of the distal spread of cancer. Ho...

Metastasis is the primary cause of death in non-hematological cancers yet there are no specific therapeutics against it because of a lack of validated targets. The Weinberg lab demonstrated that bone-marrow derived mesenchymal stem cells (MSCs) home to the stroma of breast tumors and induce metastasis. We verified these findings in MDA-MB-231 (MDA)...

Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent micro...

Circulating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circ...

Circulating tumor cells (CTCs) are present at low concentrations in the peripheral blood of patients with solid tumors. It has been proposed that the isolation, ex vivo culture, and characterization of CTCs may provide an opportunity to noninvasively monitor the changing patterns of drug susceptibility in individual patients as their tumors acquire...

Melanoma is a highly invasive malignancy frequently presenting with blood-borne metastases, in which recent breakthroughs in targeted and immunological therapies highlight the need for both early detection of invasion and monitoring of drug responses. We adapted a microfluidic device to capture circulating tumor cells (CTCs) in patients with melano...

There is an unmet clinical need for biomarkers to identify breast cancer patients at an increased risk of developing brain metastases. The objective is to identify gene signatures and biological pathways associated with human epidermal growth factor receptor 2-positive (HER2+) brain metastasis. We combined laser capture microdissection and gene exp...

The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. A complementary...

Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, we analyzed human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT. After Snail-1 induction, epithelial m...

Insulin-like growth factor 2 (IGF2), a developmentally regulated and maternally imprinted gene, is frequently overexpressed in pediatric cancers. Although loss of imprinting (LOI) at fetal promoters contributes to increased IGF2 in tumors, the magnitude of IGF2 expression suggests the involvement of additional regulatory mechanisms. A microRNA (miR...

When resectable, invasive pancreatic ductal adenocarcinoma (PDAC) is most commonly treated with surgery and radiochemotherapy. Given the intricate local anatomy and locoregional mode of dissemination, achieving clean surgical margins can be a significant challenge. On the basis of observations that cathepsin E (CTSE) is overexpressed in PDAC and th...

Epithelial-mesenchymal transition (EMT) of adherent epithelial cells to a migratory mesenchymal state has been implicated in tumor metastasis in preclinical models. To investigate its role in human cancer, we characterized EMT in circulating tumor cells (CTCs) from breast cancer patients. Rare primary tumor cells simultaneously expressed mesenchyma...

Epithelial to mesenchymal transition (EMT) has been postulated to contribute to the migration and dissemination of cancer cells, but supporting histopathological evidence is limited. We used a microfluidic device to isolate circulating tumor cells (CTCs), combined with multiplex fluorescent RNA-in-situ hybridization (ISH) and RNA sequencing, to qua...

Androgen deprivation therapy (ADT) is initially effective in treating metastatic prostate

Unlabelled: Androgen deprivation therapy (ADT) is initially effective in treating metastatic prostate cancer, and secondary hormonal therapies are being tested to suppress androgen receptor (AR) reactivation in castration-resistant prostate cancer (CRPC). Despite variable responses to AR pathway inhibitors in CRPC, there are no reliable biomarkers...

Resistance to BRAFV600E inhibitors is associated with reactivation of mitogen-activated protein kinase (MAPK) signaling at different levels in melanoma. To identify downstream effectors of MAPK signaling that could be used as potential additional therapeutic targets for BRAFV600E inhibitors, we used hTERT/CDK4R24C/p53DD-immortalized primary human m...

Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. Although these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an end...

Current prognostic factors are poor at identifying patients at risk of disease recurrence after surgery for stage II colon cancer. Here we describe a DNA microarray-based prognostic assay using clinically relevant formalin-fixed paraffin-embedded (FFPE) samples. A gene signature was developed from a balanced set of 73 patients with recurrent diseas...

Human tumors often contain slowly proliferating cancer cells that resist treatment, but we do not know precisely how these cells arise. We show that rapidly proliferating cancer cells can divide asymmetrically to produce slowly proliferating "G0-like" progeny that are enriched following chemotherapy in breast cancer patients. Asymmetric cancer cell...

Expression of ER−alpha in the ZF-TF-transduced fulvestrant resistant cells. Control-transduced (R238) and ZF-TF transduced fulvestrant resistant cells (F7, 19, 64, 70, 83 and 115) were exposed to vehicle or fulvestrant (100 nM) for 6 days. Cells were lysed in RIPA buffer containing protease inhibitor, and 100 micrograms of each lysate was loaded on...

Chemotherapeutic agents and investigational compounds used in the drug sensitivity screen. (XLS)

Genes (72) constituting the common fulvestrant-resistant gene expression signature. (XLS)

Gene Sets enriched in ZF-TF 64-induced fulvestrant resistant cells (Gene Cluster 2: 225 genes). (XLS)

Gene Sets enriched in ZF-TF 115-induced fulvestrant resistant cells (Gene Cluster 4: 123 genes). (XLS)

Drug Sensitivity of ZF-TF-transduced fulvestrant resistant cells. Control MCF7 (R238 cells) and ZF-TF fulvestrant resistant (F7, 19, 64, 70, 83 and 115) cells (5×103) were exposed to vehicle or 20 uM of multiple selective target inhibitors (see supplemental Table 1) for 72 hrs as previously described. Cell growth/viability was quantitated by fluore...

Gene Sets enriched in ZF-TF 7, 19, and 70-induced fulvestrant resistant cells (Gene Cluster 1: 230 genes). (XLS)

Gene Sets enriched in ZF-TF 83-induced fulvestrant resistant cells (Gene Cluster 3: 135 genes). (XLS)

Gene Sets enriched in all ZF-TF-induced fulvestrant resistant cells (Gene Cluster 5: 72 genes). (XLS)