Modulation of Cell Death Pathways in Cancer Stem Cells: Targeting Histone Demethylases

Cancer stem cells (CSCs) are tumor initiating cells within the tumor mass; that play a critical role in cancer pathogenesis. CSCs regulate cancer cell survival, metastatic potential, resistance to conventional radio-chemotherapy, disease relapse and poor prognosis. Recent studies have established that the drug resistant cancers and cancer cell lines possess high stem cell like traits compared to their drug sensitive counterparts. Histone demethylases are recently been linked to drug induced reversible tolerant state in cancers. Lysine histone demethylases are enzymes those demethylate lysines in histones and can act as transcriptional repressors or activators. Apart from histones other cellular proteins like E2F1, Rb, STAT3 and p53 are also regulated by methylation and de-methylation cycles. In cancer cells these enzymes regulate cell survival, migration, invasion, and proliferation. This review summarizes the current progress of research on the role of histone demethylases in supporting drug tolerant cancer stem cell state and their potential as a drug target.


INTRODUCTION
Cancer stem cells (CSCs) represent a small population of cells existing within the tumor mass and possess many characteristics of normal stem cells [1].They can divide indefinitely, renew themselves and differentiate into various tumor forming cells.The existence of CSCs has long been debated but now presence of such rare population of cells is well documented in various cancer types [1,2].To date, the presence of cancer stem cells has been reported in acute and chronic myeloid leukemia, brain, head and neck, lung, breast, pancreatic, gastrointestinal, colon, prostate and skin cancers [3][4][5][6][7][8][9][10][11][12][13][14][15].Standard radio-therapy and chemotherapy is effective against most of the cancer cells but cancer stem cells are highly resistant to these therapies and remain viable post treatment [15][16][17].Among other cancer cells these cells are present as a very small population though, they are believed to induce tumor relapse, sometimes many years after the "successful" treatment of the primary tumors [18][19][20][21][22].There are many ways by which a cancer stem cell escapes cell death.The differentiation status of a cell is an important intrinsic factor while components of microenvironment such as secreted survival factors, adhesion-mediated apoptosis resistance and hypoxic environment are important extrinsic factors regulating CSC survival [23][24][25][26][27][28][29].CSCs are also responsible for the enhanced migratory and metastatic potential of cancer cells [2].During metastasis cancer cell detaches itself from cancer mass and resists cell death activation after detachment, a process known as anoikis [30][31][32][33].Epithelial to mesenchymal transition (EMT) is an important step in metastasis of various cancers; it is characterized by transition of a cell to a much invasive, elongated mesenchymal form from a less invasive epithelial form [2]. Recent evidences indicates that CSC state, chemo-resistance and EMT pathways are somehow linked and activation of one induces other one and vice-versa [34][35][36][37][38][39].The acquired drug tolerant stem cell state in many cancers was found reverseble once drug treatment is discontinued, indicating the role of epigenetic mechanisms in chemo-resistance and disease relapse.Various pharmacological inhibitors of epigenetic modifiers like histone deacetylases (HDACs), DNA methyltransferase (DNMTs), have shown promising results in cancer therapy [40,41].Recently, a group of epigenetic modifiers known as histone demethylases have been the focus of intense investigation for their role in carcinogenesis [42].
Amongst this group, lysine histone demethylases are protein lysine demethylases, which act on histone and non-histone proteins [43,44].Major function of this group of enzymes is to mediate epigenetic regulation of gene transcription at the chromatin level [44].Histone demethylases have important role in various cellular proc-esses like cell cycle progression, nuclear hormone mediated and NF-kB signalling, p53 regulation, transcripttional regulation of Hox genes, fetoplacental development, regulation of DNA replication and repair processes [45,46].Histone demethylases are often found to be up-regulated in lung, colon, breast, prostate cancers and retinoblastomas [47,48].Hyperactivity of histone demethylases is responsible for enhanced cancer cell survival and poor patient prognosis [47,48].
Histone methylation, a critical event in the epigenetic regulation is controlled by specific histone methyltransferases (adds methyl group to histones) and demethylases (removes methyl group) [49].Methylations of histone H4 at lysine 20 (H4K20), histone H3 at lysines 9 (H3K9) and 27 (H3K27), leads to transcriptional repression [49,50].While the methylations of lysine 4 (K4) and lysine 36 (K36) in histone H3 (H3K4, H3K36) are usually associated with transcription activation (Figure 1) [49,50].The methyl groups in H3K4 are removed by histone demethylases LSD1, KDM1b, and JARID1A-1D, leading to transcription repression, while methyl groups in H3K27 are removed by KDM6b and in H3K9 by LSD1 resulting in transcription activation [49,51,52].Histone demthylases can either activate or repress the transcriptional program by changing the histone code at the transcription site [49].Their role in activation and repression is governed by the cell type, predominant signaling pathways and the cellular microenvironment [53][54][55].Histone demethylase LSD-1, KDM5a and KDM5b are known to maintain stem cell state in normal as well as cancer stem cells and influence induction of drug resistant phenotype in cancer cells [56].Therefore, inhibition of histone demethylases could be a potent therapeutic target to inhibit cancer stem cell growth as well as to sensitize chemoresistant cells to therapy/drug induced apoptosis.

Epigenetic Regulation of Cell Death and Proliferation
Epigenetic regulation of cell death and proliferation by histone lysine demethylases is mediated mainly through repression of p21 by LSD1 and KDM5b [56].In MLL-AF9 leukemia stem cells LSD1and p21 are essential for maintaining the properties of oncogenic potential and self renewal.p21 is a cyclin-dependent kinase (cdk) inhibitor and is a key mediator of DNA damage induced p53-dependent cell cycle arrest and apoptosis [62].In leukemic cells, p21 is necessary for self-renewal of leukemia stem cells [63,64].LSD1 and KDM5b regulate mRNA expression of anti-apoptotic gene CDKN1 (p21) [58].LSD1 also regulates expression of cellular proliferation genes CCNA2 and ERBB2 by binding directly to the promoters of these genes [58].KDM5b interacts with TFAP2C and Myc to form a complex leading to transcriptional repression of p21 [56].As LSD1 represses the expression of p21, knockdown of LSD1 in MDA-MB 231 cell model decrease the occupancy of LSD1 on the p21 promoter and significantly increase in the repressive mark of methylated H3K9 on CCNA2 and ERBB2 promoter regions [58].CCNA2 encodes Cyclin A2 that functions as CDK2 kinase activator and promotes progression of cell through G1/S and G2/M phases of cell cycle [65].ERBB2 (HER2) is a member of epidermal growth factor (EGF) receptor family of receptor tyrosine kinases.It forms heterodimer with other members of EGF receptor family, stabilizing ligand binding and enhances downstream mitogen-activated protein kinase and phosphatidylinositol-3 kinase mediated downstream sig-naling pathways [66].Over-expression of cyclin A2 and ERBB2 corresponds to a drug resistant or aggressive phenotype of tumor cells [67,68].LSD1 can also be linked to the aberrant regulation of Wnt signaling pathways in cancer cells.Wnt signaling is important to maintain cancer stem cell state in various cancers [69].Treatment of colon cancer cells with LSD1 oligoamine inhibitor SL111144 led to increases in H3K4Me3, restoring expression of secreted frizzledrelated proteins 2 (SFRP2) [70].SFRP2 ia a Wnt signaling pathway antagonist and it enhances the expression of the epithelial marker E-cadherin, through inhibition of the expression of SLUG, TWIST and SNAIL [71].SNAIL, SLUG and Twist are transcription factors involved in the epithelial mesenchymal transition (EMT) program [71].KDM6b act on H3K27 and is responsible for activation of anti-apoptotic gene Bcl2 transcription in hormone dependent breast cancers [57].Apart from normal antiapoptotic functions Bcl2 is thought to be involved in resistance to conventional cancer therapies, suggesting role of decreased apoptosis may play a role in the development of cancer [72].KDM5A-mediated H3K4 demethylase activity plays an important role in maintaining the proliferative capacity of breast cancer cells through repression of tumor suppressor genes, including BRCA1 [58].Another histone lysine demethylase JARID1B leads to repression of let-7e which then increases expression of cyclin D1 [59].Cyclin D1 is a target gene of mir let-7e mediated gene regulation.JARID1B demethylase contributes to tumor cell proliferation through the epigenetic repression of a tumor suppressor miR let-7 that has been reported to be a direct regulator of RAS expression in human cells [59,73].In lung cancer patient samples, expression of RAS and let-7 showed reciprocal pattern, which has low let-7 and high RAS in cancerous cells, and high let-7 and low RAS in normal cells [74].Other targets of let-7 are some oncogenes like high mobility group A2 (HMGA2) and MYC [74,75].Histone lysine demethylase mediated epigenetic gene regulation thus can drive tumorigenisis in cancers and inhibit programmed cell death to support cancer stem cells state.

KDM5A (JARID1A) and KDM1A (LSD1) Support Drug Tolerant Cancer Stem Cell State
Apart from specific mechanism and targets involved in histone lysine demethylase mediated regulation of cell proliferation and apoptosis, various studies link lysine histone demethylases to cancer drug tolerant stem cell state [56,[79][80][81].The Settleman lab generated erlotinib resistant versions of PC9 lung cancer cells by exposing these cells to media containing increasing concentrations of erlotinib [79].This drug tolerant state could be reversed after withdrawing the drug, suggesting a epigenetic link behind this reversible erlotinib resistance [79].
In the erlotinib resistant population of PC9 cells, KDM5A expression was increased, leading to decreased global levels of H3K4me3 and H3K4me2 (Figure 3) [79].In an another study carried out by the Zhanq lab demonstrated that pharmacological inhibition LSD1 specifically kill cancer cells expressing pluripotent stem cell markers [80].LSD1 inhibition led to the decrease in proliferation of pluripotent cancer cells including teratocarcinoma, embryonic carcinoma, seminoma or embryonic stem cells that express the stem cell markers Oct4 and Sox2 [80].They also demonstrated that LSD1 inhibition has minimum growth-inhibitory effects on non-pluripotent cancer or normal somatic cells [80].Similar kind of results were also been obtained in a study carried out in MLL-AF9 Leukemia stem cells [64].The study also found that inhibition of LSD1 using tranylcypromine analogs leads to differentiation of human AML cells without affecting normal repopulating cells [81].

LSD1 Expression and Its Potential as a Therapeutic Target in Various Cancers
LSD1 expression was found altered in many cancer types [82].In lung cancer over expression and nuclear localization of LSD1 is associated with shorter overall survival of non-small cell lung cancer (NSCLC) patients [82].LSD1 disruption using siRNA or a chemical inhibitor pargyline, up-regulates epithelial marker E-cadherin and down-regulates mesenchymal markers Twist and N-Cadherin, thus suppresses proliferation, migration and invasion of A549, H460 and 293T cells [82].
In acute myeloid leukemia (AML) inhibition of LSD1 using tranylcypromine (TCP) increases H3K4me2 and expression of myeloid-differentiation-associated genes [83].In combination with all-trans-retinoic acid (ATRA) LSD1 inhibitor TCP leads to decrease in the engraftment of human AML cells in NOD-SCID γ (with interleukin-2 (IL-2) receptor γ chain deficiency) mice [83].Effect of combination was better than the effect of either drug alone [83].
In prostate cancer LSD1 play an important role in Androgen receptor signaling.Androgen Receptor signaling is essential for prostate cancer initiation and progression [84].Androgen deprivation therapy remains the standard of care for treatment of advanced prostate cancer [84].Inhibition of LSD1 using its pharmacological inhibitor namoline blocks LSD1 demethylase activity in vitro and in vivo [85,86].Inhibition of LSD1 by namoline leads to the silencing of androgen receptor (AR)-regulated gene expression and severely impairs androgen-dependent proliferation in vitro and in vivo [85].
There are mixed reports on breast cancers, LSD1 expression is very low in most of the breast cancers.In a study carried out by Wang et al. in MDA-MB 231 (ER negative, PR negative) breast cancers cells revealed that LSD1 is a negative regulator of cancer metastasis [87].LSD1 forms a complex with NuRD (LSD1/NuRD) and regulate the metastatic potential of breast cancer cells [87].NuRD complex has histone deacetylase activity.Expression of LSD1 negatively co-relates with the expression of TGF-b, as TGFb signaling pathway is critically involved in epithelial-mesenchymal transitions and tumor invasion, ectopic expression of LSD1 leads to suppression of tumor invasion and migration [87].A very high expression of LSD1 has been observed in ER negative breast cancers in a study carried out by Soyoung et al.They further knocked down LSD1 in ER negative cells using siRNA that resulted in growth retardation of breast cancer cells in vitro [88].Similar results were obtained on LSD1 inhibition by pharmacological inhibitor clorgyline or tranylcypromine [88].The potential of LSD1 as a therapeutic target in breast cancer is still obscure.
In other cancers LSD1 expression is differentially high in cancer tissue than normal tissue.This differential expression of LSD1makes it a promising target in treatment of various cancer types.

Expression of Other Histone Lysine Demethylases in Cancers and Their Potential as Therapeutic Target
Histone lysine demethylase KDM2B express differentially in various cancers [89,90].In leukemia it acts as an oncogene and its over-expression helps in leukemia development and maintenance [89].While in aggressive brain tumors KDM2B expression is low and it imparts a negative effect on cell size and cell proliferation [90].In bladder cancers FGF-2 signaling leads to up-regulation of KDM2B.KDM2B up-regulation is essential for FGF-2 mediated cell proliferation, migration and angiogenesis [55].Expression of another histone demethylase KDM3A was found up-regulated in renal cell carcinoma and colon cancer [91].KDM3A regulates genes implicated in cancer cell growth, invasion, and survival [91].KDM3A knockdown in cells resulted in reduction of tumor growth rate in mice xenograft model [91].In nasopharyngeal carcinoma KDM3A expression negatively correlates with poor prognosis and is often found down-regulated in aggressive tumors [92].KDM4A and KDM4B were found up-regulated in prostate cancer, while KDM4B expression was high in renal carcinoma [93,94].High expression of KDM4C is associated with oncogenic pro-gression in esophageal squamous cell carcinoma and metastatic lung sarcomatoid carcinoma [96].Various gene amplifications of KDM4C were observed in Breast cancer and desmoplastic medulloblastomas leading to its over-expression in both cancer types [97,98].KDM5A expression was found altered only when cancer cells are subjected to drug induced stress [79] (discussed previously) while KDM5B is over-expressed in breast cancers and plays role of an oncogene; it represses various tumor suppressor genes including BRCA1 [99,100].In prostate cancer tissues KDM5B expression was also found high compared with benign prostate samples [101].KDM6A activity in bladder cancer is very low due to inactivating mutations [102].KDM6A function as a tumor suppressor [103].KDM6B expression was found high in hodgkin's lymphoma and prostate cancer and has been reported to rise further in metastatic prostate cancer [104,105].Another demethylase KDM8 was reported to be over-expressed in breast cancers and it supports cancer cell proliferation [106].Table 1 summarizes expression of lysine histone demetylases in various cancers.Among all histone lysine demethylases LSD1, appears to be a promising target in lung cancer, prostate cancer and acute myeloid leukemia.KDM4A, KDM4B and KDM5b could possibly be therapeutic targets in prostate

LIMITATIONS
Histone lysine demethylases were discovered in last decade and their role in cancer and other biological function came into limelight only in the last 5 years.Histone methylation modulates the structure and function of chromatin [49].The balance between the methylation and demethylation of specific histone residues at gene locus is critical for regulating gene expression.Still much is not known about the targets of these enzymes, also their role in various cancers is yet to be delineated.Current literature limits only to their potential role in few malignancies and in some cancer models to drug tolerant cancer stem cell state.Prior to the therapeutic targeting, their precise role in initiation, progression and spread/ metastasis must be deciphered.Therefore more thorough insight is required to their biology in cancer and stem cells.

FUTURE PROSPECTIVES
Histone demethylases work as erasures of histone code and they also regulate function of various proteins by lysine demethylation [43,44].This group of enzyme is known to regulate various cancer hallmarks; they participate in self renewal, differentiation, anti-apoptotic, migratory, and angiogenic pathways [45,46].Histone deacetylases another group of epigenetic enzymes function as erasures of lysine acetyl group at histones [107].HDACs are also known to modulate various cancer hallmarks and thus are an established group of therapeutic targets [107].Like HDACs, demthylases also modulate functions and expression of versatile targets and this makes them a good choice for development of cancer therapeutics.There are various HDAC inhibitors currently under cancer clinical trials [107,108].HDACs removes acetyl group from histones and make DNA more tightly packed around histones, and thus leads to transcription repression (similar to demethylases) of genes present at target site [108].Like histone demethylases, HDACs also modulate proteins non-epigenetically; p53, E2F1 and STAT3 are common targets [108].Histone demethylase inhibitors can be used in combination with the HDAC inhibitors in cancer where both HDAC and demethylase activities are high.HDAC inhibition usually leads to E2F1 and p53 activation and STAT3 inactivation [108].Therefore combination of both could be an effective way to treat cancers.A recent study using HDAC inhibitor vorinostat in combination with LSD1 inhibitor PCI-24781, reported that the combination induced a synergistic apoptotic cell death in glioblastoma multiforme cells [109].A similar study in breast cancer used HDAC inhibitor in combination with LSD1 and LSD2 inhibitors, and found a synergistic effect only in HDAC-LSD1 inhibitor combination [110].Few speculations can be made out of these studies: 1) HDAC inhibittors and Lysine demethylase inhibitors could result into a synergestic combination; and 2) Probably not all combination will be highly effective.KDM5A inhibitors could also be used in combination with HDAC inhibitors or with standard therapeutic agents to sensitize cancer cells to death.As histone lysine demethylases LSD1 and KDM5A play an important role in cancer stem cell state and chemo-resistance their inhibitors could be a promising therapy [79][80][81].
Histone lysine demethylases inhibitors could be used in combination with inhibitors of the pathways upregulated in cancer and responsible for cancer stem cell state and chemoresistance.Inhibitors of various cancer and cancer stem cell related pathways, notch, receptor tyrosine kinase, hedgehog, wnt-β-catenin and mTOR can be used in combination with each other and also with the inhibitors of histone demethylases [111][112][113][114].

CONCLUSION
The limited numbers of studies on histone lysine demethylases so far are suggestive of their potential as an attractive drug target in cancer.Histone lysine demethylases in some cases act as oncogenes and support cancer growth and progression, while other cases act as tumor suppressors and inhibit tumor growth.Most important aspect of their function relate to cancer cell quiescence or maintenance of cancer stem cell state and drug resistance.Combination of histone demethylase inhibitors with traditional cancer therapy holds a huge potential for treatment of various malignancies.However a thorough mechanistic understanding of the basic biology of histone lysine demethylases is imperative in gaining a better idea about their function in normal cellular processes as well as carcinogenesis.

ACKNOWLEDGEMENTS
We wish to apologize to all colleagues whose work, because of lack of space, could not be cited.We thank all the members of the DP Mishra

Figure 2 .
Figure 2. General role of lysine histone demethylases in maintaining cancer stem cell state.Cancer stem cells over-express histone lysine demethylases and low expression is associated with differentiated state.KDM-lysine demethylase.
laboratory for helpful discussions.This work was supported by the grants from the Defense Research and Development Organization (GAP-0058) to DP Mishra.Ravi Thakur acknowledges the support by the junior research fellowship from the Council of Scientific and Industrial Research, New Delhi.The Communication Number of this manuscript is 8325.