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Immunosuppression in the tumour microenvironment (TME) attenuates antitumor immunity, consequently hindering protective immunosurveillance and preventing effective antitumor immunity induced by cancer immunotherapy. Multiple mechanisms including immune checkpoint molecules, such as CTLA-4, PD-1, and LAG-3, and immunosuppressive cells are involved in the immunosuppression in the TME. Regulatory T (Treg) cells, a population of immunosuppressive cells, play an important role in inhibiting antitumor immunity. Therefore, Treg cells in the TME correlate with an unfavourable prognosis in various cancer types. Thus, Treg cell is considered to become a promising target for cancer immunotherapy. Elucidating Treg cell functions in cancer patients is therefore crucial for developing optimal Treg cell-targeted immunotherapy. Here, we describe Treg cell functions and phenotypes in the TME from the perspective of Treg cell-targeted immunotherapy.

Sequence of qPCR primers and sgRNAs

This file contains the results of studies with DOX-treated (i) BT-549 cells expressing a tet-CshRNA or tet-MUC1shRNA (S1A-S1D), (ii) MDA-MB-231 cells expressing a tet-CshRNA (S1E), and (iii) SUM-159 cells expressing a tet-MUC1shRNA (S1F).

This file contains ChIP qPCR primer pairs for the GAPDH and PD-L1 promoters.

Supplementary Figures S1-S11

The immune checkpoint ligand PD-L1 and the transmembrane mucin MUC1 are upregulated in triple-negative breast cancer (TNBC), where they contribute to its aggressive pathogenesis. Here, we report that genetic or pharmacological targeting of the oncogenic MUC1 subunit MUC1-C is sufficient to suppress PD-L1 expression in TNBC cells. Mechanistic investigations showed that MUC1-C acted to elevate PD-L1 transcription by recruitment of MYC and NF-κB p65 to the PD-L1 promoter. In an immunocompetent model of TNBC in which Eo771/MUC1-C cells were engrafted into MUC1 transgenic mice, we showed that targeting MUC1-C associated with PD-L1 suppression, increases in tumor-infiltrating CD8+ T cells and tumor cell killing. MUC1 expression in TNBCs also correlated inversely with CD8, CD69, and GZMB, and downregulation of these markers associated with decreased survival. Taken together, our findings show how MUC1 contributes to immune escape in TNBC, and they offer a rationale to target MUC1-C as a novel immunotherapeutic approach for TNBC treatment.Significance: These findings show how upregulation of the transmembrane mucin MUC1 contributes to immune escape in an aggressive form of breast cancer, with potential implications for a novel immunotherapeutic approach. Cancer Res; 78(1); 205–15. ©2017 AACR.

The oncogenic MUC1-C protein is overexpressed in triple-negative breast cancer (TNBC) cells and contributes to their epigenetic reprogramming and chemoresistance. Here we show that targeting MUC1-C genetically or pharmacologically with the GO-203 inhibitor, which blocks MUC1-C nuclear localization, induced DNA double-strand breaks and potentiated cisplatin (CDDP)-induced DNA damage and death. MUC1-C regulated nuclear localization of the polycomb group proteins BMI1 and EZH2, which formed complexes with PARP1 during the DNA damage response. Targeting MUC1-C downregulated BMI1-induced H2A ubiquitylation, EZH2-driven H3K27 trimethylation, and activation of PARP1. As a result, treatment with GO-203 synergistically sensitized both mutant and wild-type BRCA1 TNBC cells to the PARP inhibitor olaparib. These findings uncover a role for MUC1-C in the regulation of PARP1 and identify a therapeutic strategy for enhancing the effectiveness of PARP inhibitors against TNBC.Significance:These findings demonstrate that targeting MUC1-C disrupts epigenetics of the PARP1 complex, inhibits PARP1 activity, and is synergistic with olaparib in TNBC cells.

Supplementary Methods for detailed screening methods and the materials used.

Figure S1 Preparation experiments for CRIPSR screening. Figure S2 Quality control of the result of CRISPR screening. Figure S3 Validation experiments with PC9 or HCC827 cells transduced sgRNAs targeting ufmylation. Figure S4 ER stress pathway response involve in the phenotypic change of the PC9 cells in the abcense of ufmylation. Figure S5 Inflammation pathway activation affect the status of cancer cells through modifying the apoptosis pathway. Figure S6 Bcl-xL dependency in the cells with increased ER stress

An increasingly recognized component of resistance to tyrosine kinase inhibitors (TKI) involves persistence of a drug-tolerant subpopulation of cancer cells that survive despite effective eradication of the majority of the cell population. Multiple groups have demonstrated that these drug-tolerant persister cells undergo transcriptional adaptation via an epigenetic state change that promotes cell survival. Because this mode of TKI drug tolerance appears to involve transcriptional addiction to specific genes and pathways, we hypothesized that systematic functional screening of EGFR TKI/transcriptional inhibitor combination therapy would yield important mechanistic insights and alternative drug escape pathways. We therefore performed a genome-wide CRISPR/Cas9 enhancer/suppressor screen in EGFR-dependent lung cancer PC9 cells treated with erlotinib + THZ1 (CDK7/12 inhibitor) combination therapy, a combination previously shown to suppress drug-tolerant cells in this setting. As expected, suppression of multiple genes associated with transcriptional complexes (EP300, CREBBP, and MED1) enhanced erlotinib/THZ1 synergy. Unexpectedly, we uncovered nearly every component of the recently described ufmylation pathway in the synergy suppressor group. Loss of ufmylation did not affect canonical downstream EGFR signaling. Instead, absence of this pathway triggered a protective unfolded protein response associated with STING upregulation, promoting protumorigenic inflammatory signaling but also unique dependence on Bcl-xL. These data reveal that dysregulation of ufmylation and ER stress comprise a previously unrecognized TKI drug tolerance pathway that engages survival signaling, with potentially important therapeutic implications.Significance: These findings reveal a novel function of the recently described ufmylation pathway, an ER stress survival signaling in drug-tolerant persister cells, which has important biological and therapeutic implications. Cancer Res; 78(4); 1044–57. ©2017 AACR.

Dysregulation of the tumor-intrinsic epigenetic circuit is a key driver event for the development of cancer. Accumulating evidence suggests that epigenetic and/or genetic drivers stimulate intrinsic oncogenic pathways as well as extrinsic factors that modulate the immune system. These modulations indeed shape the tumor microenvironment (TME), allowing pro-oncogenic factors to become oncogenic, thereby contributing to cancer development and progression. Here we review the epigenetic dysregulation arising in cancer cells that disseminates throughout the TME and beyond. Recent CRISPR screening has elucidated key epigenetic drivers that play important roles in the proliferation of cancer cells (intrinsic) and inhibition of antitumor immunity (extrinsic), which lead to the development and progression of cancer. These epigenetic players can serve as promising targets for cancer therapy as a dual (two-in-one)-targeted approach. Considering the interplay between cancer and the immune system as a key determinant of immunotherapy, we discuss a novel lineage-tracing technology that enables longitudinal monitoring of cancer and immune phenotypic heterogeneity and fate paths during cancer development, progression, and therapeutic interventions.

Diffuse glioma is the most frequent and malignant neuroepithelial tumor in the central nervous system. IDH mutations are the most frequent genetic mutations in adult gliomas. The WHO Classification of Tumors, 5th edition, volume 6: Central Nervous System Tumours revised in 2021, defines the presence of IDH mutations in gliomas as astrocytoma IDH-mutant and oligodendroglioma IDH-mutant and 1p/19q-codeleted. In addition, more than 90% of IDH mutations found in gliomas are IDH1 R132H mutation. Several clinical trials of mutant IDH1 inhibitors targeting the IDH1 R132H mutation have shown their efficacy, but it has also been reported that IDH1 inhibitors alone may not provide sufficient tumor suppression. In this study, we performed genome-wide knockout screening using CRISPR/Cas9 genome editing technology to search for new drug targets that are synergistic with the mutant IDH1 inhibitor DS-1001b. The results of CRISPR screening and RNA sequence analysis revealed that several metabolism-related genes are factors that define resistance to mutant IDH inhibitor. In a tumor suppression study, treatment of two IDH1 R132H mutant glioma cell lines (MGG152 and BT142) with mutant IDH1 inhibitor suppressed production of the oncometabolite D2-HG, but did not significantly alter cell growth. However, when mutant IDH1 inhibitor were combined with inhibitor for metabolism-related genes, cell survival was significantly reduced, and the effects were synergistic. In this study, we found that administration of mutant IDH inhibitors to IDH mutant gliomas significantly altered intratumor metabolism using various molecular approaches. We then identified targets that, when combined with mutant IDH inhibitor, provide synergistic tumor suppression. These results are expected to provide a new therapeutic approach for a subset of patients who have difficulty responding to mutant IDH1 inhibitors.

BackgroundImmune checkpoint blockade (ICB) induces durable clinical responses in patients with various types of cancer. However, its limited clinical efficacy requires the development of better approaches. In addition to immune checkpoint molecules, tumor-infiltrating immunosuppressive cells including regulatory T cells (Tregs) play crucial roles in the immune suppressive tumor microenvironment. While phosphatidylinositol 3-kinase (PI3K) inhibition as a Treg-targeted treatment has been implicated in animal models, its effects on human Tregs and on the potential impairment of effector T cells are required to be clarified for successful cancer immunotherapy.MethodsThe impact of a selective-PI3K inhibitor ZSTK474 with or without anti-programmed cell death 1 (PD-1) monoclonal antibody on Tregs and CD8+T cells were examined with in vivo animal models and in vitro experiments with antigen specific and non-specific fashions using peripheral blood from healthy individuals and cancer patients. Phenotypes and functions of Tregs and effector T cells were examined with comprehensive gene and protein expression assays.ResultsImproved antitumor effects by the PI3K inhibitor in combination with ICB, particularly PD-1 blockade, were observed in mice and humans. Although administration of the PI3K inhibitor at higher doses impaired activation of CD8+T cells as well as Tregs, the optimization (doses and timing) of this combination treatment selectively decreased intratumoral Tregs, resulting in increased tumor antigen-specific CD8+T cells in the treated mice. Moreover, on the administration of the PI3K inhibitor with the optimal dose for selectively deleting Tregs, PI3K signaling was inhibited not only in Tregs but also in activated CD8+T cells, leading to the enhanced generation of tumor antigen-specific memory CD8+T cells which contributed to durable antitumor immunity. These opposing outcomes between Tregs and CD8+T cells were attributed to the high degree of dependence on T cell signaling in the former but not in the latter.ConclusionsPI3K inhibitor in the combination with ICB with the optimized protocol fine-tuned T cell activation signaling for antitumor immunity via decreasing Tregs and optimizing memory CD8+T cell responses, illustrating a promising combination therapy.

Hematopoiesis was considered a hierarchical stepwise process but was revised to a continuous process following single-cell RNA sequencing. However, the uncertainty or fluctuation of single-cell transcriptome dynamics during differentiation was not considered, and the dendritic cell (DC) pathway in the lymphoid context remains unclear. Here, we identify human B-plasmacytoid DC (pDC) bifurcation as large fluctuating transcriptome dynamics in the putative B/NK progenitor region by dry and wet methods. By converting splicing kinetics into diffusion dynamics in a deep generative model, our original computational methodology reveals strong fluctuation at B/pDC bifurcation in IL-7Rα

Most human tumors are composed of genetically and phenotypically heterogeneous cancer cell populations that poses a major challenge for the clinical management of cancer patients. Advances of single cell technologies have allowed the profiling of tumors at unprecedented depth, which in combination with newly-developed computational tools enable the dissection of tumor evolution with increasing precision. However, our understanding of mechanisms that regulate intratumoral heterogeneity and our ability to modulate it has been lagging behind. Recent data demonstrate that epigenetic regulators, including histone demethylases, may control the cell-to-cell variability of transcriptomes and chromatin profiles and they may modulate therapeutic responses via this function. Thus, the therapeutic targeting of epigenetic enzymes may be used to decrease intratumoral cellular heterogeneity and decrease treatment resistance when used in combination with other types of agents.

The oncogenic MUC1-C protein is overexpressed in triple-negative breast cancer (TNBC) cells and contributes to their epigenetic reprogramming and chemoresistance. Here we show that targeting MUC1-C genetically or pharmacologically with the GO-203 inhibitor, which blocks MUC1-C nuclear localization, induced DNA double-strand breaks and potentiated cisplatin (CDDP)-induced DNA damage and death. MUC1-C regulated nuclear localization of the polycomb group proteins BMI1 and EZH2, which formed complexes with PARP1 during the DNA damage response. Targeting MUC1-C downregulated BMI1-induced H2A ubiquitylation, EZH2-driven H3K27 trimethylation, and activation of PARP1. As a result, treatment with GO-203 synergistically sensitized both mutant and wild-type BRCA1 TNBC cells to the PARP inhibitor olaparib. These findings uncover a role for MUC1-C in the regulation of PARP1 and identify a therapeutic strategy for enhancing the effectiveness of PARP inhibitors against TNBC. Significance: These findings demonstrate that targeting MUC1-C disrupts epigenetics of the PARP1 complex, inhibits PARP1 activity, and is synergistic with olaparib in TNBC cells.

The extent of genetic and epigenetic diversity between and within patient tumors is being mapped in ever more detail. It is clear that cancer is an evolutionary process in which tumor cell intrinsic and extrinsic forces shape clonal selection. The pre-clinical oncology pipeline uses model systems of human cancer - including mouse models, cell lines, patient-derived organoids and patient-derived xenografts - to study tumor biology and assess the efficacy of putative therapeutic agents. Model systems cannot completely replicate the environment of human tumors and, even within the same cancer model, data are often irreproducible between laboratories. One hypothesis is that ongoing evolutionary processes remain relevant in laboratory models, leading to divergence over time. In a recent edition of Nature, Ben-David and colleagues showed that different stocks of widely used cancer cell lines - a staple of cancer research over many decades - are highly heterogeneous in terms of their genetics, transcriptomics and responses to therapies. The authors find compelling evidence of positive selection based on ongoing mutational processes and chromosomal instability. Thus, the origin, culture conditions and cumulative number of population doublings of cell lines likely influence experimental outcomes. Here, we summarize the key findings of this important study and discuss the practical implications of this work for researchers using cell lines in the laboratory.

An increasingly recognized component of resistance to tyrosine kinase inhibitors (TKI) involves persistence of a drug-tolerant subpopulation of cancer cells that survive despite effective eradication of the majority of the cell population. Multiple groups have demonstrated that these drug-tolerant persister cells undergo transcriptional adaptation via an epigenetic state change that promotes cell survival. Because this mode of TKI drug tolerance appears to involve transcriptional addiction to specific genes and pathways, we hypothesized that systematic functional screening of EGFR TKI/transcriptional inhibitor combination therapy would yield important mechanistic insights and alternative drug escape pathways. We therefore performed a genome-wide CRISPR/Cas9 enhancer/suppressor screen in EGFR-dependent lung cancer PC9 cells treated with erlotinib + THZ1 (CDK7/12 inhibitor) combination therapy, a combination previously shown to suppress drug-tolerant cells in this setting. As expected, suppression of multiple genes associated with transcriptional complexes (EP300, CREBBP, and MED1) enhanced erlotinib/THZ1 synergy. Unexpectedly, we uncovered nearly every component of the recently described ufmylation pathway in the synergy suppressor group. Loss of ufmylation did not affect canonical downstream EGFR signaling. Instead, absence of this pathway triggered a protective unfolded protein response associated with STING upregulation, promoting protumorigenic inflammatory signaling but also unique dependence on Bcl-xL. These data reveal that dysregulation of ufmylation and ER stress comprise a previously unrecognized TKI drug tolerance pathway that engages survival signaling, with potentially important therapeutic implications. Significance: These findings reveal a novel function of the recently described ufmylation pathway, an ER stress survival signaling in drug-tolerant persister cells, which has important biological and therapeutic implications. Cancer Res; 78(4); 1044–57. ©2017 AACR.

The immune checkpoint ligand PD-L1 and the transmembrane mucin MUC1 are upregulated in triple-negative breast cancer (TNBC), where they contribute to its aggressive pathogenesis. Here, we report that genetic or pharmacological targeting of the oncogenic MUC1 subunit MUC1-C is sufficient to suppress PD-L1 expression in TNBC cells. Mechanistic investigations showed that MUC1-C acted to elevate PD-L1 transcription by recruitment of MYC and NF-κB p65 to the PD-L1 promoter. In an immunocompetent model of TNBC in which Eo771/MUC1-C cells were engrafted into MUC1 transgenic mice, we showed that targeting MUC1-C associated with PD-L1 suppression, increases in tumor-infiltrating CD8+ T cells and tumor cell killing. MUC1 expression in TNBCs also correlated inversely with CD8, CD69, and GZMB, and downregulation of these markers associated with decreased survival. Taken together, our findings show how MUC1 contributes to immune escape in TNBC, and they offer a rationale to target MUC1-C as a novel immunotherapeutic approach for TNBC treatment. Significance: These findings show how upregulation of the transmembrane mucin MUC1 contributes to immune escape in an aggressive form of breast cancer, with potential implications for a novel immunotherapeutic approach. Cancer Res; 78(1); 205–15. ©2017 AACR.

B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) is a component of the polycomb repressive complex 1 (PRC1) complex that is overexpressed in breast and other cancers, and promotes self-renewal of cancer stem-like cells. The oncogenic mucin 1 (MUC1) C-terminal (MUC1-C) subunit is similarly overexpressed in human carcinoma cells and has been linked to their self-renewal. There is no known relationship between MUC1-C and BMI1 in cancer. The present studies demonstrate that MUC1-C drives BMI1 transcription by a MYC-dependent mechanism in breast and other cancer cells. In addition, we show that MUC1-C blocks miR-200c-mediated downregulation of BMI1 expression. The functional significance of this MUC1-C→BMI1 pathway is supported by the demonstration that targeting MUC1-C suppresses BMI1-induced ubiquitylation of H2A and thereby derepresses homeobox HOXC5 and HOXC13 gene expression. Notably, our results further show that MUC1-C binds directly to BMI1 and promotes occupancy of BMI1 on the CDKN2A promoter. In concert with BMI1-induced repression of the p16INK4a tumor suppressor, we found that targeting MUC1-C is associated with induction of p16INK4a expression. In support of these results, analysis of three gene expresssion data sets demonstrated highly significant correlations between MUC1-C and BMI1 in breast cancers. These findings uncover a previously unrecognized role for MUC1-C in driving BMI1 expression and in directly interacting with this stem cell factor, linking MUC1-C with function of the PRC1 in epigenetic gene silencing.

Mammary epithelial progenitors are the normal cell-of-origin of breast cancer. We previously defined a population of p27+ quiescent hormone-responsive progenitor cells in the normal human breast whose frequency associates with breast cancer risk. Here, we describe that deletion of the Cdkn1b gene encoding the p27 cyclin-dependent kinase inhibitor in the estrogen-induced mammary tumor-susceptible ACI rat strain leads to a decrease in the relative frequencies of Cd49b+ mammary luminal epithelial progenitors and pregnancy-related differentiation. We show by comprehensive gene expression profiling of purified progenitor and differentiated mammary epithelial cell populations that p27 deletion has the most pronounced effects on luminal progenitors. Cdkn1b-/- females have decreased fertility, but rats that are able to get pregnant had normal litter size and were able to nurse their pups implying that loss of p27 in ACI rats does not completely abrogate ovarian function and lactation. Reciprocal mammary gland transplantation experiments indicate that the p27-loss-induced changes in mammary epithelial cells are not only caused by alterations in their intrinsic properties, but are likely due to altered hormonal signaling triggered by the perturbed systemic endocrine environment observed in Cdkn1b-/- females. We also observed a decrease in the frequency of mammary epithelial cells positive for progesterone receptor (Pr) and FoxA1, known direct transcriptional targets of the estrogen receptor (Erα), and an increase in phospho-Stat5 positive cells commonly induced by prolactin (Prl). Characterization of genome-wide Pr chromatin binding revealed distinct binding patterns in mammary epithelial cells of Cdkn1b+/+ and Cdkn1b-/- females and enrichment in genes with known roles in Notch, ErbB, leptin, and Erα signaling and regulation of G1-S transition. Our data support a role for p27 in regulating the pool size of hormone-responsive luminal progenitors that could impact breast cancer risk., Author summary The frequency and proliferation of tissue-specific stem and progenitor cells is associated with the risk of malignancy. Thus, regulators of the pool size and proliferation of progenitor cells determine cancer risk. p27 is a key regulator of cellular proliferation and is required for the terminal differentiation of a number of cell types. Here we show that genetic deletion of Cdkn1b in ACI rats susceptible to estrogen-induced mammary tumors decreases the relative fraction of Cd49b+ luminal progenitors identifying p27 as a key regulator of the proliferation and pool size of these cells. Progesterone, acting via the progesterone receptor (Pr), is an important regulator of mammary epithelial cell proliferation and differentiation. Based on ChIP-seq we found that Pr targets different sets of genes in Cdkn1b+/+ and Cdkn1b-/- mammary epithelium and that this is associated with differences in proliferation and differentiation states. Thus, p27 regulates breast cancer risk and tumor development via regulating the pool size and hormonal-responsiveness of luminal progenitors.

Summary Members of the KDM5 histone H3 lysine 4 demethylase family are associated with therapeutic resistance, including endocrine resistance in breast cancer, but the underlying mechanism is poorly defined. Here we show that genetic deletion of KDM5A/B or inhibition of KDM5 activity increases sensitivity to anti-estrogens by modulating estrogen receptor (ER) signaling and by decreasing cellular transcriptomic heterogeneity. Higher KDM5B expression levels are associated with higher transcriptomic heterogeneity and poor prognosis in ER+ breast tumors. Single-cell RNA sequencing, cellular barcoding, and mathematical modeling demonstrate that endocrine resistance is due to selection for pre-existing genetically distinct cells, while KDM5 inhibitor resistance is acquired. Our findings highlight the importance of cellular phenotypic heterogeneity in therapeutic resistance and identify KDM5A/B as key regulators of this process.

We have previously reported that promoter polymorphism of myocardin-related transcription factor A (MRTF-A) is associated with coronary atherosclerosis. However, the contribution of MRTF-A to the development of atherosclerosis remains unknown. Macrophages are known to be important mediators of atherosclerosis. It has been demonstrated that local proliferation and survival of macrophages are atherogenic. In this study, we found that MRTF-A was highly expressed in lesional macrophages in human carotid atherosclerotic plaque. We then investigated the role of macrophagic MRTF-A in the pathogenesis of atherosclerosis. ApoE null MRTF-A transgenic mice (ApoE−/−/MRTF-Atg/+), in which human MRTF-A was specifically overexpressed in monocytes/macrophages, were established and fed with normal diet to examine the progression of atherosclerosis. We found that ApoE−/−/MRTF-Atg/+ aggravated atherosclerosis and lesional macrophages were more prominently accumulated in the aortic sinus of ApoE−/−/MRTF-Atg/+ than in that of ApoE−/− littermates. We also found that MRTF-A promoted proliferation and mitigated apoptosis of macrophages both in vitro and in vivo, and down regulated the expression of cyclin-dependent kinase inhibitors. From these findings, we conclude that MRTF-A modulates functional properties of pro-atherogenic macrophages. Our study may play a valuable role in understanding the pathological role of macrophagic MRTF-A in the progression of atherosclerosis.

The androgen receptor (AR) plays an essential role in prostate cancer, and suppression of its signaling with androgen deprivation therapy (ADT) has been the mainstay of treatment for metastatic hormone-sensitive prostate cancer for more than 70 y. Chemotherapy has been reserved for metastatic castration-resistant prostate cancer (mCRPC). The Eastern Cooperative Oncology Group-led trial E3805: ChemoHormonal Therapy Versus Androgen Ablation Randomized Trial for Extensive Disease in Prostate Cancer (CHAARTED) showed that the addition of docetaxel to ADT prolonged overall survival compared with ADT alone in patients with metastatic hormone-sensitive prostate cancer. This finding suggests that there is an interaction between AR signaling activity and docetaxel sensitivity. Here we demonstrate that the prostate cancer cell lines LNCaP and LAPC4 display markedly different sensitivity to docetaxel with AR activation, and RNA-seq analysis of these cell lines identified KDM5D (lysine-specific demethylase 5D) encoded on the Y chromosome as a potential mediator of this sensitivity. Knocking down KDM5D expression in LNCaP leads to docetaxel resistance in the presence of dihydrotestosterone. KDM5D physically interacts with AR in the nucleus, and regulates its transcriptional activity by demethylating H3K4me3 active transcriptional marks. Attenuating KDM5D expression dysregulates AR signaling, resulting in docetaxel insensitivity. KDM5D deletion was also observed in the LNCaP-derived CRPC cell line 104R2, which displayed docetaxel insensitivity with AR activation, unlike parental LNCaP. Dataset analysis from the Oncomine database revealed significantly decreased KDM5D expression in CRPC and poorer prognosis with low KDM5D expression. Taking these data together, this work indicates that KDM5D modulates the AR axis and that this is associated with altered docetaxel sensitivity.

BET bromodomain inhibitors (BBDIs) are candidate therapeutic agents for triple-negative breast cancer (TNBC) and other cancer types, but inherent and acquired resistance to BBDIs limits their potential clinical use. Using CRISPR and small-molecule inhibitor screens combined with comprehensive molecular profiling of BBDI response and resistance, we identified synthetic lethal interactions with BBDIs and genes that, when deleted, confer resistance. We observed synergy with regulators of cell cycle progression, YAP, AXL, and SRC signaling, and chemotherapeutic agents. We also uncovered functional similarities and differences among BRD2, BRD4, and BRD7. Although deletion of BRD2 enhances sensitivity to BBDIs, BRD7 loss leads to gain of TEAD-YAP chromatin binding and luminal features associated with BBDI resistance. Single-cell RNA-seq, ATAC-seq, and cellular barcoding analysis of BBDI responses in sensitive and resistant cell lines highlight significant heterogeneity among samples and demonstrate that BBDI resistance can be pre-existing or acquired.

Myoepithelial cells play key roles in normal mammary gland development and in limiting pre-invasive to invasive breast tumor progression, yet their differentiation and perturbation in ductal carcinoma in situ (DCIS) are poorly understood. Here, we investigated myoepithelial cells in normal breast tissues of BRCA1 and BRCA2 germline mutation carriers and in non-carrier controls, and in sporadic DCIS. We found that in the normal breast of non-carriers, myoepithelial cells frequently co-express the p63 and TCF7 transcription factors and that p63 and TCF7 show overlapping chromatin peaks associated with differentiated myoepithelium-specific genes. In contrast, in normal breast tissues of BRCA1 mutation carriers the frequency of p63+TCF7+ myoepithelial cells is significantly decreased and p63 and TCF7 chromatin peaks do not overlap. These myoepithelial perturbations in normal breast tissues of BRCA1 germline mutation carriers may play a role in their higher risk of breast cancer. The fraction of p63+TCF7+ myoepithelial cells is also significantly decreased in DCIS, which may be associated with invasive progression., Myoepithelial cells prevent tumour growth and invasion in DCIS. Here, the authors show that p63 and TCF7 cooperate to regulate a transcription factor network for the maintenance of normal myoepithelial function and altered expression of these genes perturb myoepithelial function in DCIS to promote invasive progression.

Glioma is the most common primary tumor of the central nervous system, accounting for ∼30%, and is classified by the World Health Organization (WHO) into 4 clinical grades, from I to IV. The most aggressive form of glioma is glioblastoma multiforme (GBM), with a 5-year survival rate of ∼8%.1,2 Surgical resectability is the most important prognostic factor, as effects of additional chemotherapy and radiotherapy are limited. Temozolomide (TMZ) is an alkylating agent used in the treatment of malignant gliomas, including GBM.3 In a study of 573 patients with newly diagnosed, histologically confirmed GBM randomly assigned to receive radiotherapy alone or radiotherapy plus continuous daily TMZ, statistically significant survival benefits were shown in patients given TMZ.4 However, the prognosis for most patients with GBM remains dismal, with a median survival of only 14.6 months.4 A greater understanding of the biological mechanisms for GBM oncogenesis will contribute to the development of targeted therapies that can improve patient outcome. The genome-wide analysis performed by The Cancer Genome Atlas has shown that the most frequent genetic abbreviations were identified in the signaling pathways involving receptor tyrosine kinase, phosphatidylinositol-3 kinase (PI3K), p53, and retinoblastoma protein.5 In addition, it has been suggested that GBM can be classified into 3 subgroups: proneuronal, proliferative, and mesenchymal.6 Although various targeted molecular agents have been used either as single agents or in combination therapy for GBM, few have been reported to be effective in phase II trials so far.7 Therefore, identification of new molecular targets is still of paramount importance. The cancer stem cell hypothesis proposes that tumors are driven by subpopulations of tumor cells with stem cell–like properties.8 Cancer stem cells have been isolated in multiple tumor types, including GBM. Several molecules, such as cluster of differentiation (CD )133, sex determining region Y–box 2 (Sox2), CD15, integrin-α6, and the L1 cell adhesion molecule, have been proposed as markers for cancer-initiating cells.9–16 Especially, by promoter analysis for CD133, the pathway of epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)–ERK has been shown to be involved in CD133 gene expression through Ets-family transcription factors.17 Considering the evaluation for their prognostic value, none of them was proven to be clinically useful in large-scale studies. Given the heterogeneity of GBM, further investigations are necessary to identify the treatment-resistant cell population, as these occasionally overlap with cancer-initiating cell properties. It is essential to develop tailored treatments to target this population with increased tumorigenic potential.18 In this study, through single subcutaneous passage in mice, we have developed highly aggressive variants of human GBM cell lines LN443 and U373, which showed increased tumor growth, colony-forming potential, sphere-forming potential, and invasiveness compared with the parental cell lines. Using DNA microarray analysis, we identified a novel molecular mechanism for the pathogenicity of GBM and explored new therapeutic agents that can be used for this disease.

Cancer is initiated by the transformation of stem cells or progenitor cells via a dedifferentiation process that leads to cancer stem cells; however, the process involves the activation of growth-promoting oncogenes and the inactivation of growth-constraining tumor suppressor genes. The introduction of defined factors, such as those encoded by c- Myc, Sox2, Oct3/4 and Klf4, in normal somatic cells results in their dedifferentiation into induced pluripotent stem (iPS) cells. We previously reported that these defined factors induced the development of induced multipotent cancer (iPC) cells from gastrointestinal cancer cells by reducing tumor aggressiveness. Previous studies indicated that although reprogramming may be facilitated by p53 inhibition, gain- of-function oncogenic mutations in p53 and oncogenic mutations in Kras- stimulated tumorigenic activity, and their roles in vivo are imperfectly understood. Hence, in the present study, the effect of direct injection of a Sendai virus (SeV) vector encoding four defined factors in vivo was studied using various backgrounds of transgenic and knockout mice, and was compared with that of direct injection of microRNAs (miRNAs) diluted with cationic lipid. The in vivo imaging data revealed transformation hot spots forp53 deficiency or conditional activation of mutant Kras, and the sizes were concordant with those in immuno-deficient NOD/SCID and uPA-NOG mice, as well as larger compared with those in the control mice. Overall, the present data on in vivo reprogramming indicated that Kras activation may facilitate the effect of cellular reprogramming in normal liver cells, and the effect of Kras activation is more apparent than that of tumor suppressor p53 deficiency. The results also revealed that immunodeficiency may increase the effect of reprogramming, presumably by blocking the immunosurveillance of transformed cells. These findings provide a rationale for further studies to develop a therapeutic approach involving direct in vivo reprogramming.

Cancer is initiated by the transformation of stem cells or progenitor cells via a dedifferentiation process that leads to cancer stem cells; however, the process involves the activation of growth-promoting oncogenes and the inactivation of growth-constraining tumor suppressor genes. The introduction of defined factors, such as those encoded by c-Myc, Sox2, Oct3/4 and Klf4, in normal somatic cells results in their dedifferentiation into induced pluripotent stem (iPS) cells. We previously reported that these defined factors induced the development of induced multipotent cancer (iPC) cells from gastrointestinal cancer cells by reducing tumor aggressiveness. Previous studies indicated that although reprogramming may be facilitated by p53 inhibition, gain-of-function oncogenic mutations in p53 and oncogenic mutations in Kras-stimulated tumorigenic activity, and their roles in vivo are imperfectly understood. Hence, in the present study, the effect of direct injection of a Sendai virus (SeV) vector encoding four defined factors in vivo was studied using various backgrounds of transgenic and knockout mice, and was compared with that of direct injection of microRNAs (miRNAs) diluted with cationic lipid. The in vivo imaging data revealed transformation hot spots for p53 deficiency or conditional activation of mutant Kras, and the sizes were concordant with those in immuno-deficient NOD/SCID and uPA-NOG mice, as well as larger compared with those in the control mice. Overall, the present data on in vivo reprogramming indicated that Kras activation may facilitate the effect of cellular reprogramming in normal liver cells, and the effect of Kras activation is more apparent than that of tumor suppressor p53 deficiency. The results also revealed that immunodeficiency may increase the effect of reprogramming, presumably by blocking the immunosurveillance of transformed cells. These findings provide a rationale for further studies to develop a therapeutic approach involving direct in vivo reprogramming.

The transcription factor Atonal homolog 1 (Atoh1) plays crucial roles in the differentiation of intestinal epithelium cells. Although we have reported that the Atoh1 protein was degraded in colon cancer by aberrant Wnt signaling, a recent study has indicated that the Atoh1 protein is expressed in mucinous colon cancer (MC) and signet ring cell carcinoma (SRCC). However, the roles of the Atoh1 protein in MC are unknown. To mimic MC, a mutated Atoh1 protein was stably expressed in undifferentiated colon cancer cells. Microarray analysis revealed the acquisition of not only the differentiated cell form, but also malignant potential by Atoh1 protein stabilization. In particular, Atoh1 enhanced Wnt signaling, resulting in the induction of Lgr5 as a representative stem cell marker with the enrichment of cancer stem cells. Moreover, the fluorescent ubiquitination-based cell cycle indicator system with time-lapse live imaging demonstrated cell cycle arrest in the G0/G1 phase by Atoh1 protein stabilization. In conclusion, the Atoh1 protein regulates malignant potential rather than the differentiation phenotype of MC, suggesting the mechanism by which MC and SRCC are more malignant than non-mucinous adenocarcinoma.

Objectives: This study sought to ascertain the relationship of 9p21 locus with: 1) angiographic coronary artery disease (CAD) burden; and 2) myocardial infarction (MI) in individuals with underlying CAD. Background: Chromosome 9p21 variants have been robustly associated with coronary heart disease, but questions remain on the mechanism of risk, specifically whether the locus contributes to coronary atheroma burden or plaque instability. Methods: We established a collaboration of 21 studies consisting of 33,673 subjects with information on both CAD (clinical or angiographic) and MI status along with 9p21 genotype. Tabular data are provided for each cohort on the presence and burden of angiographic CAD, MI cases with underlying CAD, and the diabetic status of all subjects. Results: We first confirmed an association between 9p21 and CAD with angiographically defined cases and control subjects (pooled odds ratio [OR]: 1.31, 95% confidence interval [CI]: 1.20 to 1.43). Among subjects with angiographic CAD (n = 20,987), random-effects model identified an association with multivessel CAD, compared with those with single-vessel disease (OR: 1.10, 95% CI: 1.04 to 1.17)/copy of risk allele). Genotypic models showed an OR of 1.15, 95% CI: 1.04 to 1.26 for heterozygous carrier and OR: 1.23, 95% CI: 1.08 to 1.39 for homozygous carrier. Finally, there was no significant association between 9p21 and prevalent MI when both cases (n = 17,791) and control subjects (n = 15,882) had underlying CAD (OR: 0.99, 95% CI: 0.95 to 1.03)/risk allele. Conclusions: The 9p21 locus shows convincing association with greater burden of CAD but not with MI in the presence of underlying CAD. This adds further weight to the hypothesis that 9p21 locus primarily mediates an atherosclerotic phenotype.

Aberrant expression of myeloid cell leukemia-1 (MCL-1) is a major cause of drug resistance in triple-negative breast cancer (TNBC) cells. Mucin 1 (MUC1) is a heterodimeric oncoprotein that is aberrantly overexpressed in most TNBC. The present studies show that targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in TNBC cells with silencing or pharmacologic inhibition with GO-203 is associated with downregulation of MCL-1 levels. Targeting MUC1-C suppresses the MEK → ERK and PI3K → AKT pathways, and in turn destabilizes MCL-1. The small molecules ABT-737 and ABT-263 target BCL-2, BCL-XL and BCL-w, but not MCL-1. We show that treatment with ABT-737 increases reactive oxygen species and thereby MUC1-C expression. In this way, MUC1-C is upregulated in TNBC cells resistant to ABT-737 or ABT-263. We also demonstrate that MUC1-C is necessary for the resistance-associated increases in MCL-1 levels. Significantly, combining GO-203 with ABT-737 is synergistic in inhibiting survival of parental and drug resistant TNBC cells. These findings indicate that targeting MUC1-C is a potential strategy for reversing MCL-1-mediated resistance in TNBC.

Cancer stem cells (CSCs), which make up only a small proportion of heterogeneous tumor cells, may possess greater ability to maintain tumorigenesis than do other tumor cell types. Breast cancer tissue is reported to contain cancer stem-like cells. In order to eradicate tumor cells, various approaches have been taken to identify the critical molecules and signaling pathways in breast CSCs. Recent findings suggest that inflammatory signaling pathways are important for the maintenance of breast CSCs. Here, we review the current understanding of the role of inflammatory pathways in these cells and discuss future perspectives of the research on and the possibility of targeting the molecules involved in these pathways for developing treatments for breast cancer.

Aims Z-band alternatively spliced PDZ-motif protein (ZASP)/Cypher is a Z-disc component of which several dilated cardiomyopathy (DCM)-associated mutations have been reported. Most of the mutations were found in exons 4 and 10 of ZASP/Cypher gene LDB3 and both exons were expressed preferentially in the heart. The aim of this study was to investigate the functional alteration of ZASP/Cypher caused by the DCM-associated mutations. Methods and results The yeast-two-hybrid method was used to identify the protein bound to a domain encoded by exon 4 of LDB3 . Interaction of ZASP/Cypher with the binding protein was investigated in relation to the functional alterations caused by LDB3 mutations. Localization of the ZASP/Cypher-binding protein was examined at the cellular level in rat cardiomyocytes. Phosphoglucomutase 1 (PGM1), a metabolic enzyme involved in glycolysis and gluconeogenesis, was identified as a protein interacting with ZASP/Cypher. PGM1 bound to ZASP/Cypher at the domains encoded by exons 4 and 10. Two LDB3 mutations in exon 4 (Ser189Leu and Thr206Ile) and another mutation in exon 10 (Ile345Met) reduced the binding to PGM1. PGM1 showed diffuse localization in the cytoplasm of rat cardiomyocytes under standard culture conditions, and distribution at the Z-discs was observed under stressed culture conditions. Binding of endogenous PGM1 and ZASP/Cypher was found to be enhanced by stress in rat cardiomyocytes. Conclusion ZASP/Cypher anchors PGM1 to Z-disc under conditions of stress. The impaired binding of PGM1 to ZASP/Cypher might be involved in the pathogenesis of DCM.

Coronary artery disease (CAD) has become a major health problem in many countries because of its increasing prevalence and high mortality. Recently, an association of a functional sequence variation, -8CG, in the human proteasome subunit alpha type 6 gene (PSMA6) with the susceptibility to CAD was reported. To validate the association, we investigated a total of 1330 cases and 2554 controls from Japanese and Korean populations for PSMA6 genotypes, and no evidence of the association was obtained in both Japanese (odds ratio (OR)=1.03, 95% confidence interval (CI); 0.90-1.19, P=0.66, allele count model) and Korean populations (OR=1.00, 95% CI; 0.86-1.17, P=0.95, allele count model). However, when a meta-analysis of data from this study and previously reported six replication studies was done, OR was 1.08 for the G allele (95% CI; 1.02-1.14, P=0.0057), suggesting that the contribution of PSMA6 to CAD was not large enough to be readily replicated. Further studies are required to establish the contribution of this variant in the susceptibility to CAD.

Hypertrophic cardiomyopathy (HCM) is a cardiac disease characterized by left ventricular hypertrophy with diastolic dysfunction. Molecular genetic studies have revealed that HCM is caused by mutations in genes for sarcomere/Z-band components including titin/connectin and its associate proteins. However, disease-causing mutations can be found in about half of the patients, suggesting that other disease-causing genes remain to be identified. To explore a novel disease gene, we searched for obscurin gene (OBSCN) mutations in HCM patients, because obscurin interacts with titin/connectin. Two linked variants, Arg4344Gln and Ala4484Thr, were identified in a patient and functional analyses demonstrated that Arg4344Gln affected binding of obscurin to Z9-Z10 domains of titin/connectin, whereas Ala4484Thr did not. Myc-tagged obscurin showed that Arg4344Gln impaired obscurin localization to Z-band. These observations suggest that the obscurin abnormality may be involved in the pathogenesis of HCM.

Dilated cardiomyopathy (DCM) is a cardiac disease characterized by dilated ventricle and systolic dysfunction. Most of the DCM patients are sporadic cases, but a certain population of DCM patients can be familial cases caused by mutations in genes for sarcomere/Z-disc components including titin/connectin. However, disease-causing mutations could be identified only in a part of the familial DCM patients, suggesting that there should be other disease causing genes for DCM. To explore a novel disease gene for DCM, we searched for mutations in FHL2, encoding for four and half LIM protein 2 (FHL2) in DCM patients, because FHL2 is known to associate with titin/connectin. A missense mutation, Gly48Ser, was identified in a patient with familial DCM. Functional analysis demonstrated that the FHL2 mutation affected the binding to titin/connectin. Because FHL2 protein is known to tether metabolic enzymes to titin/connectin, these observations suggest that the Gly48Ser mutation may be involved in the pathogenesis of DCM via impaired recruitment of metabolic enzymes to the sarcomere.

The rhesus macaque exhibits individual differences in susceptibility and resistance to infectious agents such as simian immunodeficiency virus (SIV) under experimental conditions, and these may be genetically determined at least in part by major histocompatibility complex (MHC) class I polymorphism. Although the importance of defining MHC class I polymorphism is well recognized, development of a generic and comprehensive molecular typing method of MHC class I alleles of the rhesus macaque has been hampered because, during the evolution of this species, multiple copies of similar DNA sequences have been generated by duplication events including the coding sequences of Mamu-A and Mamu-B loci. We report here a newly developed reference strand-mediated conformation analysis (RSCA)-based typing method of multiple Mamu-A and Mamu-B cDNAs that allowed us to estimate the number of expressed alleles. This technique detected 1-7 Mamu-A signals and 2-12 Mamu-B signals in a single sample, indicating that the number of functional alleles may vary. By comparing the data from the parents with those from the descendants in the breeding colony, several MHC class I haplotypes consisting of variable numbers of functional Mamu-A and Mamu-B alleles could be assigned.

Coronary artery disease (CAD) and stroke are the major health problems in many countries because of their increasing prevalence and high mortality. It is well known that CAD and stroke are based on atherosclerosis and shared environmental and genetic risk factors. Recently, an association of a functional sequence variation −154G>A in the angiotensin receptor-like 1 (AGTRL1) with a susceptibility to stroke was reported. In this study, we investigated a total of 1479 CAD cases and 2062 controls from the Japanese and Korean populations to validate the association of AGTRL1 with CAD. However, we obtained no evidence of the association in both the Japanese (odds ratio (OR)=0.95, 95% confidence interval (CI); 0.82–1.10, P=0.47, allele count model) and Korean (OR=0.90, 95% CI; 0.77–1.05, P=0.18, allele count model) populations. In addition, there was no trend of association between the risk allele and severity of coronary atherosclerosis. These data suggested that AGTRL1 did not contribute much to the atherosclerosis of the coronary artery.

The rs1333049 (G/C) polymorphism located on chromosome 9p21.3 is a candidate to influence extreme longevity owing to its association with age-related diseases, notably coronary artery disease (CAD). We compared allele/genotype distributions of rs1333049 in cases (centenarians) and controls (younger adults, without (healthy) or with CAD) in two independent cohorts: Spanish (centenarians: n = 152, 128 women, 100–111 years; healthy controls: n = 343, 212 women, age

Breast cancer is one of the most common cancers in humans. However, our understanding of the cellular and molecular mechanisms underlying tumorigenesis in breast tissues is limited. Here, we identified a molecular mechanism that controls the ability of breast cancer cells to form multicellular spheroids (mammospheres). We found that heregulin (HRG), a ligand for ErbB3, induced mammosphere formation of a breast cancer stem cell (BCSC)–enriched population as well as in breast cancer cell lines. HRG-induced mammosphere formation was reduced by treatment with inhibitors for phosphatidyl inositol 3-kinase (PI3K) or NF-κB and by expression of IκBα-Super Repressor (IκBαSR), a dominant-negative inhibitor for NF-κB. Moreover, the overexpression of IκBαSR in breast cancer cells inhibited tumorigenesis in NOD/SCID mice. Furthermore, we found that the expression of IL8, a regulator of self-renewal in BCSC-enriched populations, was induced by HRG through the activation of the PI3K/NF-κB pathway. These findings illustrate that HRG/ErbB3 signaling appears to maintain mammosphere formation through a PI3K/NF-κB pathway in human breast cancer.

A deletion/insertion (Del/Ins) polymorphism of 28 base pairs (bp) in the 3′ untranslated region (UTR) of fibrinogen alpha gene (FGA) was associated with thromboembolic diseases, but the underlying mechanisms remain unknown. Computational predication reveals that the 28 bp polymorphic fragment is complementary to the sequence of a microRNA, miR-759. In this study, we aim to investigate the association and implicated mechanisms between FGA polymorphisms and the susceptibility to chronic thromboembolic pulmonary hypertension (CTEPH). The Del/Ins polymorphism was analyzed in 190 patients with CTEPH and 628 controls. The FGA 3′UTR and miR-759 interaction was investigated using luciferase assay and quantitative RT-PCR method. Expression of miR-759 and FGA in human tissues was investigated by RT-PCR. The results reveal that the allele frequency of Ins was significantly higher in the patients than in the controls (55.8 vs. 47.1%, P = 0.003, odds ratio = 1.42, 95% confidence interval: 1.13–1.79). Both miR-759 and FGA were expressed in human liver. Co-transfection of miR-759 decreased the expression and mRNA stability of reporter gene containing the FGA 3′UTR. The effect of miR-759 was stronger on the Ins allele than on the Del allele. These observations suggest that the expression of FGA was regulated by miR-759 through its interaction at the polymorphic 3′UTR sequence, which was associated with the susceptibility to CTEPH.