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Targeting oral cancer epigenome via LSD1

Oral squamous cell carcinoma (OSCC) accounts for the
majority of head and neck cancers. Treatment for OSCC
frequently comprises a combination of surgery, radio-
therapy, and chemotherapy. However, resistance to
therapy complicates treatment, and the 5-year survival
rate remains at ~65 percent. Understanding contributors
to disease progression and treatment resistance is
needed to promote patient outcomes. Lysine-specific
demethylase 1 (LSD1) is an amine oxidase with de-
methylase activity and has been implicated in maintain-
ing the undifferentiated state of cancer-initiating cells.
We have determined that lysine-specific demethylase 1
(LSD1) promote growth and metastasis of human and
mouse OSCC [1, 2].
LSD1 expression is elevated in clinical OSCC
compared to dysplastic and hyperplastic tissue speci-
mens, and is absent in adjacent normal tissues. In a
tissue microarray containing a diverse population of 80
OSCC (different grade/stage) of the larynx, tongue, and
submandibular gland, LSD1 staining positively
correlated with disease grade. Indeed, bioinformatics
analysis of mRNA expression data from The Cancer
Genome Atlas (TCGA) (from more than 300 OSCC)
confirmed that LSD1 expression increases with tumor
stage and grade.
We have established OSCC mouse models for
understanding the basic mechanism and therapeutic pre-
clinical applications [1-5]. We found that LSD1
knockdown in implanted HSC-3 orthotopic tumors
attenuates tumor growth and metastasis, whereas over-
expression of LSD1 promotes disease progression.
Further, small molecule inhibitors (e.g., GSK-LSD1) of
LSD1 attenuate disease progression, EGFR-induced
signaling, and tumor-promoting gene expression
(MMP13, LOXL4, and CTGF) in patient-derived
xenografts. Microarrays followed by gene set enrich-
ment analysis also showed that GSK-LSD1 inhibits key
mediators of OSCC [1].
LSD1 has a dual and context-dependent role in
transcriptional regulation Notch signaling [6] and
regulation of androgen receptor in prostate cancer [7].
LSD1 can demethylate H3K4 during gene repression and
H3K9 during gene activation. LSD1 demethylates his-
tone and non-histone genes by removing mono- and di-
methyl groups from histone H3 at lysine 4 (H3K4me1/2)
without affecting trimethylation. Inactivation of LSD1
promotes G1 arrest and induces differentiation-specific
genes by selectively modulating the methylation states
of H3K4 and H3K9. Thus, expression of LSD1 may
enable epigenetic regulation of its targets, and
identifying LSD1-regulated molecular signaling
mechanisms could reveal new targets for OSCC
therapy. We recently implicated LSD1 in a novel action
with the Hippo signaling effector Yes-Associated
Protein (YAP), EGFR, NF-kB, and epithelial-mesen-
chymal transition in OSCC. GSK-LSD1 blocks YAP-
induced oncogenic signaling pathways in patient-
derived tonsillar epithelial, myoepithelial, and osteo-
sarcoma tumor cells. YAP and TAZ, key effectors of
the Hippo pathway, drive pro-tumorigenic signals in
OSCC [5]. GSK-LSD1 inhibits YAP-activated down-
stream targets such as SERPINE1 and CTGF in micro-
array analysis. However, which genes are functional
targets of LSD1 and exert the effects on these signaling
pathways—ultimately promoting OSCC progression
and metastasis—is not known.
EGFR signaling is implicated in OSCC progression
metastasis and therapeutic resistance. Inhibition of
LSD1 attenuates proliferation and EGF-induced signal-
ing, phospho-AKT, and ERK1/2 as well as NF-kB and
its associated transcriptional networks. Further, GSK-
LSD1 attenuates NF-kB signaling, which is implicated
in inflammatory signaling pathways and checkpoint
regulation. Although GSK-LSD1 attenuates NF-KB
signaling, whether it attenuates downstream inflamma-
tory effectors is not known.
Taken together, these results led us to ask the following
questions (Figure 1): Is LSD1 a key mediator of the pro-
inflammatory response in OSCC? Does LSD1 regulate
OSCC-specific mechanisms, and if so, can it be
exploited for monotherapy or combination therapies?
Our long-term objectives are to understand the LSD1-
induced molecular mechanism(s) underlying OSCC
growth and metastasis and to identify its therapeutic
potential for this disease. Epigenetic inhibitors that
could reset the OSCC epigenome may synergistically
correct deleterious effects induced by somatic mutations
or oncogenic pathways. Currently, various LSD1
inhibitors, including GSK-LSD1 (GlaxoSmithKline)
[8], are in phase 1 clinical trials for other cancer. LSD1
induced novel OSCC specific mechanisms, chemical
and biologic inhibitors under development to interfere
aberrant biological function of LSD1 could be applied
to OSCC therapy. Epigenetic targetting of OSCC via
ManishV.Bais  AGING2017,Vol.9,No. 12    2455 AGING
LSD1 may have broader implications for other tumor
types and translational studies.
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inducedbyEGFR‐ orYAPinducedsignalingpathways.LSD1
therebyinhibitOSCC.Unknownrolesareindicatedby(?).    2456AGING
... However, the tumor cell based on studies present an opposite data. In oral squamous cell carcinoma inhibition of LSD1 with GSK-LSD1 attenuated NF-κB signaling, which was implicated with inflammatory signalling pathways and checkpoint regulation [39,40]. Kim et al. went further, claiming that phosphorylation of LSD1 is a key epigenetic factor for the amplification of the inflammatory response. ...
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Background/aims: Inflammation is the body's natural response to stress in the broadest sense. The regulatory mechanisms that control this process, some of which are still unclear, are needed to balance the immune response, but also when insufficient, can cause immunodeficiency resulting in infection, cancer, neurodegeneration or other serious disorders. In this study, we focused on defining the role of lysine-specific demethylase 1 (LSD1), an enzyme involved in modulating the methylation state of lysine, including histone and non-histone proteins, in shaping the inflammatory profile of endothelial cells. Methods: To determine the role of LSD1 in the inflammatory response of ECs, cells were stimulated with lipopolysaccharide (100 ng/ml LPS) in the presence and absence of an LSD1 inhibitor (2-PCPA). A transcription model of LSD1 deficient cells (HMEC-1 LSD1 KD) obtained by lentiviral shRNA transduction was also used. The indicated cellular models were analyzed by gene profiling, monitoring of p65 shuttling by Western blotting and immunofluorescence staining. Also chromatin immunoprecipitation (ChIP) was performed to identify the interactions between selected: IL-6/p65 and LSD1. Results: Analysis of both experimental models revealed an altered inflammatory response following both LSD1 inhibition and LSD1 silencing. We observed decreased U-937 monocytes recruitment to LPS-activated endothelial cells and decreased extracellular secretion of many proinflammatory cytokines, also confirmed at the transcript level by RT-qPCR. Monitoring of the LPS-induced p65 translocation revealed inhibition of the NF-kB subunit in LSD1 KD vs nonT as well as due to pretreatment of 2-PCPA cells. Gene profiling performed with RNA microarrays confirmed the obtained biochemical data at the transcript level. Conclusion: In conclusion, the conducted studies showed a proinflammatory profile of LSD1 activity in endothelial cells, revealed by the inhibition of the enzyme activity and confirmed at the transcriptional level by the inhibition of its expression. Although we found significant changes in the modification of interactions between monocytes and endothelial cells as well as in cytokine/chemokine release and expression that were consistent with the altered NF-κB-p65 translocation into the nucleus, we did not identify a direct interaction between LSD1 and the transcription factor. Our finding may have important implications for prevention of cardiovascular diseases at their first stage - activation of the endothelium as well as for tumor cell biology, providing evidence for the use of LSD1 inhibitors to reduce the inflammatory response, which enhances tumor tissue remodeling, angiogenesis and metastasis.
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Controversy always existed on the utility of chemically induced mouse mammary carcinogenesis models as valid equivalents for the study of human breast cancer. Here, we performed whole exome and RNA sequencing on long latency mammary tumors (218 ± 27 days) induced by the carcinogen 7,12-Dimethylbenzathracene (DMBA) and short latency tumors (65 ± 11 days) induced by the progestin Medroxyprogesterone Acetate (MPA) plus DMBA in CD2F1 mice. Long latency tumors displayed a high frequency of Pi3kca and/or Pten mutations detected in 11 of 13 (85%) long latency cases (14/22, 64% overall). Eighty-two percent (9/11) of tumors carried the Pik3ca H1047L/R hot-spot mutation, as frequently found in human breast cancer. These tumors were luminal-like and mostly ER/PR+, as in humans. Transcriptome profiling indicated a significant activation of the PI3K-Akt pathway (p=3.82e-6). On the other hand MPA+DMBA induced short latency tumors displayed mutations in cancer drivers not commonly found mutated in human breast cancer (e.g. Hras and Apc). These tumors were mostly basal-like and MPA exposure led to Rankl overexpression (60 fold induction) and immunosuppressive gene expression signatures. In summary, long latency DMBA induced mouse mammary tumors reproduce the molecular profile of human luminal breast carcinomas representing an excellent preclinical model for the testing of PIK3CA/Akt/mTOR pathway inhibitory therapies and a good platform for the developing of additional preclinical tools such as syngeneic transplants in immunocompetent hosts.
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Unlabelled: Over 45,000 new cases of oral and pharyngeal cancers are diagnosed and account for over 8,000 deaths a year in the United States. An environmental chemical receptor, the aryl hydrocarbon receptor (AhR), has previously been implicated in oral squamous cell carcinoma (OSCC) initiation as well as in normal tissue-specific stem cell self-renewal. These previous studies inspired the hypothesis that the AhR plays a role in both the acquisition and progression of OSCC, as well as in the formation and maintenance of cancer stem-like cells. To test this hypothesis, AhR activity in two oral squamous cell lines was modulated with AhR prototypic, environmental and bacterial AhR ligands, AhR-specific inhibitors, and phenotypic, genomic and functional characteristics were evaluated. The data demonstrate that: (i) primary OSCC tissue expresses elevated levels of nuclear AhR as compared with normal tissue, (ii) AhR mRNA expression is upregulated in 320 primary OSCCs, (iii) AhR hyperactivation with several ligands, including environmental and bacterial ligands, significantly increases AhR activity, ALDH1 activity, and accelerates cell migration, (iv) AhR inhibition blocks the rapid migration of OSCC cells and reduces cell chemoresistance, (v) AhR knockdown inhibits tumorsphere formation in low adherence conditions, and (vi) AhR knockdown inhibits tumor growth and increases overall survival in vivo These data demonstrate that the AhR plays an important role in development and progression of OSCC, and specifically cancer stem-like cells. Prototypic, environmental, and bacterial AhR ligands may exacerbate OSCC by enhancing expression of these properties. Implications: This study, for the first time, demonstrates the ability of diverse AhR ligands to regulate AhR activity in oral squamous cell carcinoma cells, as well as regulate several important characteristics of oral cancer stem cells, in vivo and in vitro Mol Cancer Res; 14(8); 696-706. ©2016 AACR.
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Oral squamous cell carcinoma (OSCC) is a prevalent form of cancer that develops from the epithelium of the oral cavity. OSCC is on the rise worldwide, and death rates associated with the disease are particularly high. Despite progress in understanding of the mutational and expression landscape associated with OSCC, advances in deciphering these alterations for the development of therapeutic strategies have been limited. Further insight into the molecular cues that contribute to OSCC is therefore required. Here we show that the transcriptional regulators YAP (YAP1) and TAZ (WWTR1), which are key effectors of the Hippo pathway, drive pro-tumorigenic signals in OSCC. Regions of pre-malignant oral tissues exhibit aberrant nuclear YAP accumulation, suggesting that dysregulated YAP activity contributes to the onset of OSCC. Supporting this premise, we determined that nuclear YAP and TAZ activity drives OSCC cell proliferation, survival, and migration in vitro, and is required for OSCC tumor growth and metastasis in vivo. Global gene expression profiles associated with YAP and TAZ knockdown revealed changes in the control of gene expression implicated in pro-tumorigenic signaling, including those required for cell cycle progression and survival. Notably, the transcriptional signature regulated by YAP and TAZ significantly correlates with gene expression changes occurring in human OSCCs identified by "The Cancer Genome Atlas" (TCGA), emphasizing a central role for YAP and TAZ in OSCC biology. This study defines a YAP/TAZ-regulated transcriptional program in OSCC, and reveals novel roles for nuclear YAP/TAZ activity in the onset and progression of this cancer. Copyright © 2015, American Association for Cancer Research.
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Graphical Abstract Highlights d LSD1 is broadly associated with AR-regulated enhancers, and a subset is H3T6ph marked d LSD1 is associated with CoREST at these sites and interacts with FOXA1 d Despite coactivator function at these sites, LSD1 mediates their H3K4 demethylation d LSD1 coactivation is through demethylation of novel histone or nonhistone substrates In Brief Cai et al. show that lysine-specific demethylase 1 (LSD1), although generally a transcriptional corepressor through H3K4 demethylation, functions broadly as a coactivator for androgen receptor and interacts with FOXA1 on androgen-stimulated genes. LSD1-mediated H3K4 demethylation persists at these sites, indicating a distinct coactivator function mediated by demethylation of other substrates.
The lysyl oxidase propeptide (LOX-PP) is derived from pro-lysyl oxidase (Pro-LOX) by extracellular biosynthetic proteolysis. LOX-PP inhibits breast and prostate cancer xenograft tumor growth and has tumor suppressor activity. Although, several intracellular targets and molecular mechanisms of action of LOX-PP have been identified, LOX-PP uptake pathways have not been reported. Here we demonstrate that the major uptake pathway for recombinant LOX-PP (rLOX-PP) is PI3K-dependent macropinocytosis in PWR-1E, PC3, SCC9, MDA-MB-231 cell lines. A secondary pathway appears to be dynamin- and caveola dependent. The ionic properties of highly basic rLOX-PP provide buffering capacity at both high and low pHs. We suggest that the buffering capacity of rLOX-PP, which serves to limit endosomal acidification, sustains PI3K-dependent macropinocytosis in endosomes which in turn is likely to facilitate LOX-PP endosomal escape into the cytoplasm and its observed interactions with cytoplasmic targets and nuclear uptake. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Epigenetic dysregulation has emerged as an important mechanism in cancer. Alterations in epigenetic machinery have become a major focus for targeted therapies. The current report describes the discovery and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. This small molecule is a potent, selective, orally bioavailable, mechanism-based irreversible inactivator of LSD1. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) is sensitive to LSD1 inhibition. The subset of SCLC lines and primary samples that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes, suggesting this may be used as a predictive biomarker of activity. Copyright © 2015 Elsevier Inc. All rights reserved.
Activating mutations in NOTCH1, an essential regulator of T cell development, are frequently found in human T cell acute lymphoblastic leukemia (T-ALL). Despite important advances in our understanding of Notch signal transduction, the regulation of Notch functions in the nucleus remains unclear. Using immunoaffinity purification, we identified NOTCH1 nuclear partners in T-ALL cells and showed that, beyond the well-characterized core activation complex (ICN1-CSL-MAML1), NOTCH1 assembles a multifunctional complex containing the transcription coactivator AF4p12, the PBAF nucleosome remodeling complex, and the histone demethylases LSD1 and PHF8 acting through their demethylase activity to promote epigenetic modifications at Notch-target genes. Remarkably, LSD1 functions as a corepressor when associated with CSL-repressor complex and as a NOTCH1 coactivator upon Notch activation. Our work provides new insights into the molecular mechanisms that govern Notch transcriptional activity and represents glimpse into NOTCH1 interaction landscape, which will help in deciphering mechanisms of NOTCH1 functions and regulation.