Cancer Letters 374 (2016) 12–21

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

The histone demethylase LSD1 is a novel oncogene and therapeutic target in oral cancer Yanling Wang a,1, Yumin Zhu a,1, Qiong Wang a,1, Huijun Hu a,1, Zhongwu Li a,b, Dongmiao Wang b, Wei Zhang c, Bin Qi c, Jinhai Ye b, Heming Wu b, Hongbing Jiang b, Laikui Liu a,c, Jianrong Yang a,b, Jie Cheng a,* a

Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University, Jiangsu 210029, China Department of Oral and Maxillofacial Surgery, Nanjing Medical University, Jiangsu 210029, China c Department of Oral Pathology, Nanjing Medical University, Jiangsu 210029, China b

A R T I C L E

I N F O

Article history: Received 20 December 2015 Received in revised form 31 January 2016 Accepted 1 February 2016 Keywords: LSD1 Histone demethylase Epigenetic modification Oral cancer

A B S T R A C T

The histone demethylase LSD1 functions as a key pro-oncogene and attractive therapeutic target in human cancer. Here we sought to interrogate the oncogenic roles of LSD1 in OSCC tumorigenesis and therapeutic intervention by integrating chemical-induced OSCC model, genetic and pharmacological loss-offunction approaches. Our data revealed that aberrant LSD1 overexpression in OSCC was significantly associated with tumor aggressiveness and shorter overall survival. Increased abundance of LSD1 was detected along with disease progression in DMBA- or 4NQO-induced OSCC animal models. LSD1 depletion via siRNA-mediated knockdown in OSCC cells resulted in impaired cell proliferation, migration/ invasion, tumorsphere formation and reduced xenograft growth while inducing cell apoptosis and enhancing chemosensitivity to 5-FU. Moreover, treatments of LSD1 chemical inhibitors (pargyline and tranylcypromine) induced its protein reduction probably via enhanced protein degradation and produced similar phenotypic changes resembling LSD1 silencing in OSCC cells. Pharmacological inhibition of LSD1 by intraperitoneal delivery of these inhibitors resulted in impaired xenograft overgrowth. Taken together, our data reveal the tumorigenic roles of LSD1 and identified LSD1 as a novel biomarker with diagnostic and prognostic significance, and also establish that targeting LSD1 by chemical inhibitors is a viable therapeutic strategy against OSCC. © 2016 Elsevier Ireland Ltd. All rights reserved.

Introduction Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide with well-established etiologic factors including smoking abuse and excessive alcohol consumption, human papillomavirus (HPV) infection, and so on [1]. Despite considerable advances in comprehensive and multimodality therapy against this devastating disease over the past decades, however, the long-term survival rate has not been markedly improved, especially in patients with advanced lesions [2]. Locoregional relapse and cervical lymph node metastasis are the most prevalent factors which significantly affect patients’ prognosis. Although OSCC initiation and progression are intricately associated with aberrant activation of oncogenes, inactivation of tumor suppressors as well as epigenetic abnormalities, the limited and incomplete information regarding the molecular carcinogenesis of OSCC has hampered the development

* Corresponding author. Tel.: +81 25 85031880; fax: +81 25 85031880. E-mail addresses: [email protected]; [email protected] (J. Cheng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.canlet.2016.02.004 0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.

of biomarkers and therapeutic strategies with high potency and sensitivity [3]. Therefore, further unraveling the molecular mechanisms and identifying novel therapeutic targets are of great importance to improve patient prognosis. Aberrant epigenetic dysregulation including DNA methylation and histone modification is a hallmark of human cancer [4]. Molecular machinery that governs these epigenetic modifications has become a major focus for targeted therapies for years [5]. Among them, the histone lysine-specific demethylase 1 (LSD1, also known as KDM1A), initially identified in 2004 as the first histone demethylase, has been found to be a bona fide oncogene implicated in a broad spectrum of malignancies [6]. LSD1serves not only as a transcriptional repressor as a core component of CoREST or NuRD co-repressor complexes by mediating the demethylation of H3K4m1/ m2, but also as a transcriptional activator via demethylation of H3K9m1/m2 in diverse biological settings [7,8]. Mounting evidence has well established that LSD1 plays critical roles in diverse fundamental cellular processes including cell proliferation, differentiation, epithelial–mesenchymal transition and stem cell fate determination [9–12]. Moreover, LSD1 is frequently overexpressed in multiple human cancers including head and neck cancer, and is

Y. Wang et al./Cancer Letters 374 (2016) 12–21

also associated with aggressive clinicopathological features and adverse patient outcomes [13–16]. We have reported that LSD1 is aberrantly overexpressed in a majority of tongue squamous cell carcinomas (the most prevalent site for primary OSCC), and significantly associated with aggressive clinicopathological features and unfavorable prognosis [14]. More importantly, pharmacological inhibition or genetic depletion of LSD1 inhibited cancer cell proliferation, differentiation, invasion and migration, and induced tumor recession in animal models, whereas its overexpression contributed to malignant transformation through chromatin modifications in vitro and in vivo [15,17–20]. Therefore, these abovementioned findings strongly underscore the importance of LSD1 as an important oncogenic driver and cancer biomarker, and also provide evidence that inhibition of LSD1 may represent an attractive anti-cancer therapeutic approach. Previous studies have offered intriguing clues that LSD1 functions as an oncogenic driver, novel biomarker as well as a viable therapeutic target in human cancers [16,18,21]. However, its expression pattern and detailed biological roles in OSCC remain largely undefined yet. In this study, we sought to assess the LSD1 expression and functions by integrating genetic and pharmacological approaches using OSCC cell lines, animal models and tumor specimens. Our findings here further highlight that LSD1 is critically involved in OSCC tumorigenesis as well as aggressive phenotype, and hold great potential as a novel diagnostic marker and therapeutic target for OSCC. Materials and methods Detailed experimental materials, methods and relevant references were described in supplementary experimental procedures. These experiments were performed as described previously with minor modifications [22–24]. All experimental studies involving humans and animals were approved by the Research Ethic Committee and Animal Research Committee of Nanjing Medical University.

Results Overexpressed LSD1 in OSCC is associated with aggressive clinicopathological features and patient prognosis We have provided initial evidence that LSD1 is aberrantly overexpressed in a major fraction of tongue squamous cell carcinoma (the most prevalent site for OSCC), and its overexpression is associated with cancer aggressiveness and unfavorable patient prognosis [14]. To extend these findings, we further evaluated the abundance of LSD1 in OSCC cell lines and tissue samples. The realtime RT-PCR data revealed markedly increased LSD1 transcripts in all OSCC cells relative to the immortalized oral epithelial cell line (HIOCE) (Fig. 1A). The western blot results further indicated remarkably upregulated LSD1 protein in the OSCC cell lines examined (Fig. 1B). Additionally, LSD1 protein was pronouncedly elevated in fresh OSCC samples as compared to the pair-matched adjacent noncancerous tissues (n = 4) (Fig. 1C). To characterize the subcellular distribution of LSD1 in oral cancer cells, cellular immunofluorescence was performed in three selected cell lines. As shown in Fig. 1D, in line with its putative roles as an epigenetic modifier, LSD1 was readily detected and mainly identified in the nucleus. Then, we sought to measure LSD1 abundance by immunohistochemistry in an independent cohort comprising 64 primary OSCC patients. As shown in Fig. 1E, LSD1 positive staining was frequently identified in the nucleus in cancer cells, whereas weak or negative staining was detected in the normal counterparts. Based on our immunohistochemical staining scores, high LSD1 expression was identified in approximately 65.6% (42/64) in cancer samples and 25.0% (5/ 20) in normal counterparts, thus indicating aberrant LSD1 overexpression in a major fraction of OSCC (P < 0.001, Table 1). To further understand the clinical significance of LSD1 overexpression in OSCC, we next aimed to identify the potential

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Table 1 LSD1 expression pattern in OSCC and normal oral mucosa. LSD1 expression

Normal oral mucosa OSCC

p-values

Negative

Low

High

5 0

10 22

5 42

60 Tumor size T1–T2 T3–T4 Pathological grade I II–III Primary site Tongue Buccal Mouth floor Gingiva Others Cervical node metastasis N(0) N(+) Clinical stage I–II III–IV

Cases

LSD1

p-values

Low

High

64 37 27

22 14 8

42 23 19

27 37

8 14

19 23

0.7894

48 16

19 3

29 13

0.2233

34 30

9 13

25 17

0.1926

20 16 13 9 6

8 6 4 2 2

12 10 9 7 4

0.9072

37 27

17 5

20 22

0.0329

34 36

16 11

18 25

0.0346

0.5977

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Fig. 1. LSD1 is overexpressed in oral squamous cell carcinoma and is associated with patients’ survival. A: LSD1 mRNA levels were measured by real-time RT-PCR in seven OSCC cell lines as compared with the human immortalized oral epithelial cell line (HIOEC). B: LSD1 protein levels were determined by western blot (WB) in OSCC cell lines as compared to HIOEC. C: LSD1 protein abundance was measured by WB in 4 pairs of fresh OSCC and adjacent non-tumor tissues. The “N” stands for non-tumor tissue, and “C” stands for cancer in the lower panel. D: Subcellular localization of LSD1 was visualized by immunofluorescence assay in selected OSCC cell lines. E: LSD1 expression in human normal oral mucosa and OSCC specimens was evaluated by immunohistochemical staining. Scale bar: 100 μm. F: Overall survival analyses of patients with high or low expression of LSD1 based on IHC data were estimated by Kaplan–Meier method and compared with log-rank test. Data shown here are mean ± SD from two or three independent experiments. *p < 0.05, **p < 0.01, ANOVA analysis.

observed in 4-week and 10-week DMBA-treated animals. As expected, exophytic tumor-like or invasive lesions were evident in 16-week DMBA-treated animals. The histopathological analyses were further confirmed by the multiple stages induced by DMBA including epithelial hyperplasia, dysplasia/carcinoma in situ as well as invasive squamous cell carcinoma (Fig. 2A, upper panel). Overall, this model largely recapitulated the typical multiple stages of OSCC, reminiscence of human OSCC initiation and progression. Moreover, immunohistochemical staining of LSD1 in tissue samples derived from diverse stages of the OSCC model (Fig. 2A, lower panel) indicated negative/low staining in most normal epithelial and positive

staining with diverse degrees in epithelial hyperplasia, dysplasia and SCC. Furthermore, as displayed in Fig. 2B, significant LSD1 overexpression was observed in the majority of SCC samples (7/9, 77.8%), whereas much less significant overexpression was observed in samples with epithelial hyperplasia (1/8, 12.5%) and dysplasia (2/8, 25%). Importantly, similar findings about LSD1 expression in oral tumorigenesis were also observed in the 4-NQOinduced tongue SCC model (Fig. S1, data not shown). Collectively, our data support the idea that LSD1 might contribute to chemicalinduced OSCC development and function as a key oncogene driving oral tumorigenesis.

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Table 3 Univariate and multivariate survival analyses of prognostic factors for patients with OSCC. Variable

Gender (male, female) Age (≤60, >60) Tumor size (T1–T2, T3–T4) Pathological grade (I, II–III) Primary sites (tongue, bucaal, mouth floor, gingiva etc.) Cervical nodal metastasis (N0, N+) Clinical stage (I–II, III–IV) LSD1 expression (low, high)

Univariate survival analysis

Multivariate survival analysis

Hazard ratio

95% CI

p-value

Hazard ratio

95% CI

p-value

1.269 0.716 2.222 2.009 0.893 2.395 1.474 3.337

(0.575,2.801) (0.336,1.525) (0.970,5.092) (0.880,4.590) (0.650,1.227) (1.669,4.313) (0.637,3.409) (.995,11.187)

0.555 0.386 0.059 0.098 0.485 0.032* 0.364 0.051

1.736 1.160 2.406 1.739 0.884 3.247 0.539 4.236

(0.660,4.570) (0.507,2.654) (0.759,7.628) (0.616,4.906) (0.616,1.267) (0.776,4.810) (0.146,1.993) (1.170,15.342)

0.264 0.725 0.136 0.296 0.502 0.021* 0.355 0.028*

* The numbers in bold indicate statistical significance.

LSD1 promotes cell proliferation, migration/invasion, chemoresistance and cancer stem cell maintenance in OSCC Given the proposed tumorigenic functions of LSD1 in multiple cancers and our abovementioned findings of LSD1 as a novel OSCC biomarker, we next sought to dissect the oncogenic roles of LSD1 in OSCC via siRNA-mediated loss-of-function approach. Three siRNA sequences targeting human LSD1 were designed and delivered into cells (Cal27 and HN6) with relatively high levels of endogenous LSD1. The siRNA with the highest knockdown efficiency was selected and utilized in the following experiments (Fig. S2A,B). As shown in Fig. 3A, the protein abundance of LSD1 was greatly diminished after siLSD1 treatment, accompanied by a concurrent global increase of H3K4me2 in both cell lines, thus verifying the knockdown efficiency and specificity. Subsequently, the relevant phenotypic changes of cells after LSD1 knockdown were further examined in detail. Impaired cell proliferation and increased proportions of apoptotic cells were observed in LSD1 knockdown cells as measured by MTT, flow cytometry and colony formation assays (Fig. 3B–D). Moreover, LSD1 depletion induced markedly impaired migration and invasion as de-

tected via wound healing and transwell invasion assays (Fig. 3E,F). In line with these phenotypic alterations induced by LSD1 knockdown, the abundance of selected makers for cell proliferation, apoptosis and motility underwent the corresponding changes, such as increased cleaved PARP and E-cadherin, as well as diminished Cyclin D1 and vimentin (Fig. 3G). Furthermore, LSD1 knockdown also significantly enhanced the chemotherapeutic sensitivity of the cytotoxic agent 5-FU in both Cal27 and HN6 cells, although such effects were negligible for another chemotherapeutic agent, cisplatin, as gauged by cell viabilities at different time points (Fig. S2C,D). Together, these data indicate that LSD1 functions as a pro-oncogene critically involved in cell proliferation, apoptosis, invasion as well as chemotherapeutic response in OSCC cells. Several lines of evidence have uncovered the key roles of LSD1 for normal and malignant stem cell maintenance, self-renewal and fate decision [10,25–27]. Importantly, cancer stem cell or tumorinitiating cells are believed to account for tumor development, recurrence and metastasis as well as therapeutic resistance in a broad spectrum of human cancers including OSCC [28,29]. Intrigued by this, we hypothesized that LSD1 might be another critical

Fig. 2. LSD1 expression in DMBA-induced buccal SCC model. A: LSD1 abundance and localization was determined by immunohistochemistry in samples obtained from consecutive diverse SCC stages. Upper panel: HE staining; lower panel: IHC staining of LSD1. Scale bar: 100 μm. B: LSD1 expression was compared in samples derived from different SCC stages. *The p-value 0.0232 was obtained by comparing normal and other disease stages (chi-square test). If individual lesions were compared (chi-square test), the p values were 0.9658 (normal vs hyperplasia), 0.5698 (normal vs dysplasia and carcinoma in situ), 0.0048 (normal vs carcinoma), 0.5965 (hyperplasia vs dysplasia and carcinoma in situ), 0.0088 (hyperplasia vs carcinoma), 0.0513 (dysplasia and carcinoma in situ vs carcinoma).

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Fig. 3. LSD1 knockdown inhibits cell proliferation, migration and invasion, and tumorsphere formation as well as triggers apoptosis in OSCC cells. A: Endogenous LSD1 was efficiently silenced by siRNA in Cal27 and HN6 cells. Representative images of WB are shown. B: Cell viability and proliferation were remarkably suppressed when endogenous TAZ was silenced as measured by MTT assay. C: Increased percentages of cells undergoing apoptosis were evident following LSD1 knockdown as assayed by Annexin V–PI staining. D: The colony formation potential was inhibited in LSD1 depleted cells as compared to control. E,F: The migration and invasion abilities were significantly reduced in siLSD1-transfected cells in wound healing and transwell assays, respectively. G: The expression of several genes including Cyclin D1, cleaved-PARP, E-cadherin and vimentin was measured by western blot. Representative images are shown. H: Tumorsphere formation ability was significantly reduced in siLSD1-treansfected cells relative to cells with control siRNA. Scale bar: 50 μm. Data shown here are mean ± SD from three independent experiments; *p < 0.05, **p < 0.01, Student’s t-test.

regulator for stem cell-like traits in OSCC. As shown in Fig. S3A, compared to the CD44 − CD133 − subpopulation cells with limited tumorigenic potential, LSD1 protein was significantly enriched in the CD44+CD133+ subpopulation that originated from Cal27 and HN6 cell lines which were phenotypically identified as unique cells with tumor-initiating properties by in vitro tumorsphere and in vivo tumorigenic assays [30]. The tumorsphere assay as surrogate readout for CSCs-like properties was also exploited in cells with LSD1 depletion. Indeed, the number of primary and secondary tumorspheres formed from dissociated cells cultured in serum-free media clearly showed that LSD1 depletion pronouncedly impaired the abilities of tumorsphere formation (Figs. 3H and S3B), thus suggesting that LSD1 might be another novel factor in the complex regulatory network responsible for oral cancer stem cell-like properties. LSD1 is critical for the outgrowth of OSCC in vivo To further substantiate the tumorigenic roles of LSD1 in OSCC, We next developed an OSCC xenograft animal model by subcutaneous inoculation of shRNA-mediated LSD1 stable silencing cells. The tumor xenograft incidence of LSD1 knockdown and control cells during our observation period remained similar, although the latency period of LSD1-knockdown cells was significantly longer than the control counterparts (data not shown). Upon animal euthanasia, the tumor volume and weight were detected and compared. Not sur-

prisingly, our data revealed that tumor growth was substantially compromised when endogenous LSD1 was potently inhibited (Fig. 4A,B). In agreement with this, the staining of LSD1 and proliferative marker Ki67 revealed compromised cell proliferation presumably due to LSD1 knockdown (Fig. 4C). Collectively, these findings suggest that LSD1 is critical for OSCC cell proliferation and tumor growth in vivo, and has great potential as a therapeutic target for clinical translational purposes. Pharmacological inhibition of LSD1 phenocopies LSD1 knockdown in vitro The data presented thus far have revealed the essential roles of LSD1 and its therapeutic potentials in OSCC. Accumulating evidence has indicated that LSD1 can be successfully inhibited by MAO inhibitors in diverse types of cells especially cancer cells, thus highlighting the promising translational potential of LSD1 targeting by chemical compounds against cancer [17,18,21]. We therefore hypothesized that LSD1 might be therapeutically targeted by chemical inhibitors in oral cancer, especially for those with LSD1 hyperactivation. To substantiate this, we first determined whether two small-molecule LSD1 inhibitors, PG and TCP, were able to inhibit its functions in vitro. Unexpectedly, both PG and TCP exposure remarkably reduced LSD1 protein abundance in a time- and dosedependent manner in Cal27 and HN6 cells (Fig. 5A,B). Similar results

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Fig. 4. LSD1 knockdown resulted in impaired xenograft tumor growth in vivo. A: Tumor masses were formed by subcutaneous inoculation of HN6 cells with (HN6-shLSD1, right flank) or without (HN6-NC, left flank) stable LSD1 silencing in an OSCC xenograft model. The final volume and weight of the tumor xenograft were compared between these two groups. B: Tumor masses were formed by subcutaneous inoculation of Cal27 cells with (Cal27-shLSD1, right flank) or without (Ca27-NC, left flank) stable LSD1 knockdown in the OSCC xenograft model. The final volume and weight of the tumor xenograft were compared between these two groups. C: Immunohistochemical staining of LSD1 and Ki-67 was performed in tumor xenograft samples. Scale bar: 100 μm. **p < 0.01, Student’s t-test.

were also observed in another cell line, FADU (Fig. S4A). As expected, the LSD1 substrate H3K4me2 was concomitantly upregulated following PG or TCP exposure (Fig. 5A,B; lower panel). However, the LSD1 mRNA levels remained largely unaltered after PG or TCP treatment (Fig. 5C, and data not shown), suggesting these inhibitors induced LSD1 reduction probably via a post-transcriptional manner, for example, by inducing protein degradation. Indeed, the proteasome inhibitor MG132 treatment resulted in increased endogenous LSD1 (Fig. 5D) and was capable, at least in part, to abrogate the inhibitory effects of PG and TCP on LSD1 abundance (Fig. 5E). Collectively, these data suggest that PG and TCP potently inhibited endogenous LSD1 abundance presumably by inducing its degradation in OSCC cells. Next, we measured the phenotypic changes of OSCC cells following these inhibitors’ exposure. Not surprisingly, impaired cell proliferation and migration, tumorsphere formation as well as increased apoptosis were evident upon PG or TCP treatment (Figs. 6A–D and S4B). Compared with single agent treatment alone, more pronounced anti-proliferative effects were observed when cells were challenged with both PG/TCP and 5-FU (Fig. S4C). Furthermore, the expression changes of markers for cell proliferation, apoptosis and migration further confirmed the phenotypic changes upon PG or TCP exposure (Fig. 6E). Collectively, these findings support

the notion that pharmacological targeting of LSD1 by chemical inhibitors largely phenocopies LSD1 knockdown in OSCC cells. Moreover, these data further collaborate the pivotal tumorigenic roles of LSD1 underlying OSCC progression and highlight the therapeutic potential of LSD1 targeting against OSCC. Pharmacological inhibition of LSD1 inhibited OSCC outgrowth in vivo To further confirm the therapeutic effects of PG and TCP against oral cancer, we generated a xenograft animal model and treated the animals with a single agent by intraperitoneal administration. After tumor masses reached approximately 150 mm3 in volume, these animals were randomly distributed into three groups and received PG or TCP alone (150 mg/kg/day) or vehicle every day for 2 consecutive weeks. The PG and TCP treatments were well tolerated in animals as evidenced by absence of weight loss and adverse changes in blood counts or serum chemistry in drug-treated mice as compared to control animals (data not shown). As indicated in Fig. 7A, tumor volume and weight of mice that received PG or TCP treatments were much lower than those in control animals (P < 0.01). Most tumors exhibited retarded growth or regression after drug treatment. The immunohistochemical staining data further revealed remarkably diminished LSD1 and Ki67 staining as well as increased TUNEL staining in chemical-treated samples (Fig. 7B). Taken

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Fig. 5. Small-molecule inhibitor of LSD1 (PG and TCP) reduced LSD1 expression probably via inducing LSD1 protein degradation in OSCC cells. A: PG or TCP treatment resulted in LSD1 protein reduction in a dosage-dependent manner in Cal27 cells. The global H3K4me2 was also increased following PG or TCP treatment in vitro. B: PG or TCP treatment resulted in LSD1 protein reduction in a time-dependent manner in Cal27 and HN6 cells. C: LSD1 mRNA levels remain largely unaltered after PG and TCP exposure in Cal27 cells as measured by real-time RT-PCR. D: LSD1 protein abundance was significantly increased when Cal27 cells were incubated with MG132 (10 μM) for indicated times. E: The LSD1 reduction induced by PG and TCP treatments was partially abrogated by MG132 co-treatment in Cal27 cells. Cells were serum-starved for 6 h and treated with PG (5 mM), TCP (2 mM) and MG132 (10 μM) for another 12 h. After treatment, the cells were harvested and subjected to western blotting assay.

together, our data reveal that the chemical inhibitors PG and TCP have therapeutic potency against oral cancer at least partially through inhibiting LSD1.

prognosis. More importantly, depletion of LSD1 by genetic silencing and pharmacological approaches displayed potent anti-cancer effects in vitro and in vivo.

Discussion

LSD1 overexpression and its clinicopathological significance in OSCC

The past decades have highlighted the significance of aberrant epigenetic dysregulation as the key hallmark in human cancer. Such epigenetic alterations are usually reversible and dynamically regulated, thus raising the possibility that they can be therapeutically exploited as novel therapeutic targets against cancer [5]. To date, increasing evidence has linked diverse histone modifications to cancer initiation and progression [31]. Here we provide evidence that the histone demethylase LSD1 is aberrantly overexpressed in OSCC and associated with cancer aggressiveness and unfavorable

Growing evidence has indicated that LSD1 is usually upregulated and possesses oncogenic properties in diverse cancers, and is associated with advanced stages, regional or distant metastasis, as well as poor survival, thus indicative of the potential of LSD1 as a useful cancer biomarker [16,19,27]. Our previous study has also revealed an elevated LSD1 in tongue SCC with important prognostic significance [14]. Consistently, our data from OSCC cell lines and human samples indicate remarkable upregulation of LSD1 abundance in OSCC. Importantly, the immunohistochemical data from

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Fig. 6. PG and TCP treatments resulted in impaired proliferation, migration and tumorsphere formation and induced cell apoptosis in OSCC cells. A: PG and TCP treatments inhibited cell proliferation in Cal27 cells as determined by MTT assay. B: High ratio of cells undergoing apoptosis following PG and TCP challenge was detected by Annexin V–PI staining assay. C: Cell migration ability was impaired in cells treated with PG and TCP as compared to vehicle-treated cells as measured by transwell assay. D. Tumorsphere formation was significantly reduced in cells with PG and TCP as compared to control. E: The protein levels of cyclin D1, cleaved-PARP, E-cadherin and vimentin were determined when cells were individually treated PG (5 mM) and TCP (2 mM) for 48 h, respectively. Data shown here are mean ± SD from three independent experiments; *p < 0.05, **p < 0.01, Student’s t-test and ANOVA analysis.

chemical-induced OSCC animal models further revealed that LSD1 expression increased along with the disease progression from normal to hyperplasia, dysplasia and finally invasive SCC. Together, these findings clearly indicate that aberrant LSD1 overexpression represents a key pathological and molecular feature of OSCC, and also suggest that LSD1 might serve as an oncogene mediating OSCC development. Previous reports have revealed that deregulated LSD1 expression is associated with clinicopathological parameters and adverse treatment outcomes in human cancer, thus holding potential diagnostic and prognostic significance [13,16]. In accordance with these findings, our data indicate that elevated LSD1 is significantly associated with cervical node metastasis, advanced clinical stages and reduced overall survival in our patient cohort. Notably, multivariate survival analyses further identified LSD1 as an important and independent prognostic factor to predict patient survival, thus suggesting that assessment of LSD1 abundance in the OSCC sample might provide valuable information regarding patient prognosis and indication for careful follow-up management. Therefore, our data provide strong evidence that LSD1 is a novel diagnostic and prognostic biomarker for OSCC, which may be further translated and exploited in the clinic. Tumorigenic roles of LSD1 in OSCC initiation and progression The well-established clinical significance of LSD1 overexpression in human cancer strongly underscores its essential tumorigenic roles. Indeed, our findings derived from both OSCC animal models strongly suggested essential oncogenic functions of LSD1 driving OSCC tumorigenesis. This idea is supported by recent findings from Wada et al. as they reported that overexpression of LSD1 primes hematopoietic stem cells for malignant transformation, and LSD1 overexpression appears to be the first hit in T-cell leukemogenesis [32]. Moreover, our data from siRNA-mediated and pharmacological loss-of-function assays reveal that LSD1 has multiple oncogenic roles, for example regulating cell proliferation, apoptosis, migra-

tion and invasion, and chemosensitivity in oral cancer cells, which is generally in line with LSD1 roles as an oncogene. Several lines of evidence have revealed that these oncogenic functions executed by LSD1 might be associated with its partners or downstream targets such as p53, E2F1, Snail and E-cadherin in diverse physiopathological settings [11,33,34]. Very recently, when our manuscript was under preparation, Narayanan et al. identified that modulating E2F signaling was an important mechanism for LSD1 in promoting cell proliferation in oral cancer [35]. Moreover, LSD1 physically interacted with the promoter of E-cadherin and downregulated its expression via decreasing local H3K4, thus contributing to colon cancer metastasis [16]. Alternatively, LSD1 interacted with EMT master regulator Snail/Slug and in turn was recruited to their target gene promoters, resulting impaired cell migration and invasion in breast cancer [34]. Our data from in vitro cellular experiments support the idea that LSD1 functioned as an oncogene largely by modulating diverse downstream targets in cancers. Previous studies have established that LSD1 participates in the maintenance of pluripotency and balances self-renewal and differentiation in human embryonic stem cells [10]. Subsequent research further identified LSD1 as an essential regulator of cancer stem cell traits in leukemia and breast cancer [26,27]. Here we provide evidence that LSD1 might be another pivotal regulator underlying CSC stemness as gauged by the facts that LSD1 is significantly enriched in the CD44+CD+133 subpopulation with unique cancerinitiating properties, and impaired tumorsphere formation was observed upon LSD1 depletion. Complementary to this, increased cisplatin chemosensitivity was observed upon endogenous LSD1 knockdown and positive associations between LSD1 overexpression and cervical nodes metastasis in our clinical samples. Therefore, these findings provide compelling evidence that LSD1 serves as a bona fide oncogene driving OSCC initiation and progression. However, it remains an open and interesting question to further delineate the mechanism responsible for LSD1 hyperactivation and identify downstream targets mediating its functions.

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Fig. 7. Pharmacological depletion of LSD1 by PG and TCP inhibited xenograft tumor overgrowth in vivo. A: PG or TCP treatment by intraperitoneal injection inhibited tumor growth in a xenograft animal model. Final tumor volume and weight were compared in tumor-bearing animals receiving PG, TCP or vehicle. B: Representative images of H&E staining and LSD1, Ki-67 and TUNEL immunohistochemical staining in tumor xenograft samples. Scale bar: 50 μm. Data shown here are mean ± SD from two independent experiments; *p < 0.05, **p < 0.01, ANOVA analysis.

Therapeutic inhibition of LSD1 in OSCC Considering the importance of LSD1 oncogenic functions during cancer initiation and progression as well as the reversibility of epigenetic modification, targeting LSD1 by chemical inhibitors has become an attractive therapeutic strategy against cancer with LSD1 hyperactivation [21,31]. Indeed, a line of evidence has revealed that several types of small-molecule inhibitors such as monoamine oxidase inhibitor and oligoamine analogs were successfully developed to inhibit LSD1 activity and yield remarkable anti-cancer effects [17,18,36,37]. Surprisingly, we find here that TCP and PG induced LSD1 protein reduction without detectable mRNA alteration in OSCC cells in a time- and dosage-dependent manner, accompanied by a global increase of H3K4me2. This finding seems in contrast with previous studies where chemical inhibitors irreversibly inactivated LSD1 via covalent modification of its bound FAD cofactor [17,18]. We speculate that this discrepancy might be due to different cell types with diverse genetic background, and distinct treatment dosage and duration. Intrigued by unaltered LSD1 mRNA abundance following PG or TCP treatment and increased LSD1 protein after MG132 treatment, we provided further evidence that LSD1 re-

duction induced by TCP and PG was partially attributed to its reduced protein stability. However, much more definitive evidence is still needed to confirm this finding. Consistent with previous findings, both TCP and PG treatments resulted in impaired proliferation, migration, invasion, CSC maintenance and enhanced chemosensitivity in OSCC cells. More importantly, these chemicals by intraperitoneal delivery significantly retarded xenograft tumor overgrowth in vivo. In addition, these chemicals’ delivery in vivo was well tolerated as these inhibitors have been widely used as clinical antidepressant agents for decades. These findings are generally consistent with previous reports in which small-molecule LSD1 inhibitors yield significant therapeutic effects in cancer. Accumulating evidence suggests that these anticancer effects induced by LSD1 small-molecule inhibitors might be mediated by H3K4me2 or H3K9me2 gain as well as DNA methylation in a target gene promoter, which in turn modulates specific gene expression in diverse cancer contexts [17,18,38]. Notably, several bioactive small inhibitors of LSD1 with high specificity and potency have been developed to selectively target cancer cells with stem cell properties while displaying minimum growth-inhibitory effects on cancer cells with limited tumor-initiating potential or normal

Y. Wang et al./Cancer Letters 374 (2016) 12–21

somatic cells [37,39,40]. Moreover, LSD1 inhibitor TCP plus alltans-retinoic acid potently targeted leukemia-initiating cells and displayed superior effects to treatment with either drug alone in acute promyelocytic leukemia [17]. Thus, these findings confer another advantage to LSD1 inhibitors as therapeutic strategy against human cancer as they displayed high potency on the unique subpopulation-cancer stem cell which is largely responsible for cancer recurrence, metastasis and drug resistance. More efforts are warranted to develop the LSD1 inhibitor with greater specificity and potency, as well as less side-effects. In conclusion, we have revealed the tumorigenic roles of LSD1 responsible for oral cancer initiation and progression and identified LSD1 as a novel biomarker with diagnostic and prognostic significance. Our findings further establish that targeting LSD1 by chemical inhibitors is a viable therapeutic strategy against OSCC. Acknowledgements This work is financially supported, in whole or in part, by the National Natural Science Foundation of China (81572669), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (2014-37), Natural Science Foundation of Jiangsu Province (BK20151561), China Postdoctoral Science Foundation (2014M560436), Jiangsu Postdoctoral Science Foundation (1402162C), Jiangsu Creative Training Project for Graduates in Colleges (SJZZ_0119) and Jiangsu Creative Training Project for College Student (201510312053X). Conflict of interest The authors declare that they have no competing interests. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2016.02.004. References [1] A.D. Rapidis, P. Gullane, J.D. Langdon, J.L. Lefebvre, C. Scully, J.P. Shah, Major advances in the knowledge and understanding of the epidemiology, aetiopathogenesis, diagnosis, management and prognosis of oral cancer, Oral Oncol. 45 (2009) 299–300. [2] R. Prestwich, K. Dyker, M. Sen, Improving the therapeutic ratio in head and neck cancer, Lancet Oncol. 11 (2010) 512–513. [3] N. Stransky, A.M. Egloff, A.D. Tward, A.D. Kostic, K. Cibulskis, A. Sivachenko, et al., The mutational landscape of head and neck squamous cell carcinoma, Science 333 (2011) 1157–1160. [4] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144 (2011) 646–674. [5] T.K. Kelly, D.D. De Carvalho, P.A. Jones, Epigenetic modifications as therapeutic targets, Nat. Biotechnol. 28 (2010) 1069–1078. [6] Y. Shi, F. Lan, C. Matson, P. Mulligan, J.R. Whetstine, P.A. Cole, et al., Histone demethylation mediated by the nuclear amine oxidase homolog LSD1, Cell 119 (2004) 941–953. [7] M.G. Lee, C. Wynder, N. Cooch, R. Shiekhattar, An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation, Nature 437 (2005) 432–435. [8] E. Metzger, M. Wissmann, N. Yin, J.M. Muller, R. Schneider, A.H. Peters, et al., LSD1 demethylates repressive histone marks to promote androgen-receptordependent transcription, Nature 437 (2005) 436–439. [9] S. Amente, L. Lania, B. Majello, The histone LSD1 demethylase in stemness and cancer transcription programs, Biochim. Biophys. Acta 2013 (1829) 981–986. [10] A. Adamo, B. Sese, S. Boue, J. Castano, I. Paramonov, M.J. Barrero, et al., LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells, Nat. Cell Biol. 13 (2011) 652–659. [11] H. Kontaki, I. Talianidis, Lysine methylation regulates E2F1-induced cell death, Mol. Cell 39 (2010) 152–160. [12] O.G. McDonald, H. Wu, W. Timp, A. Doi, A.P. Feinberg, Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition, Nat. Struct. Mol. Biol. 18 (2011) 867–874. [13] Y. Yu, B. Wang, K. Zhang, Z. Lei, Y. Guo, H. Xiao, et al., High expression of lysine-specific demethylase 1 correlates with poor prognosis of patients with esophageal squamous cell carcinoma, Biochem. Biophys. Res. Commun. 437 (2013) 192–198.

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