Lncmapk6 Drives MAPK6 Expression and Liver TIC Self-Renewal

Huang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:105 https://doi.org/10.1186/s13046-018-0770-y

RESEARCH Open Access LncMAPK6 drives MAPK6 expression and liver TIC self-renewal Guanqun Huang1†, Hui Jiang2†, Yueming He2, Ye Lin3, Wuzheng Xia3, Yuanwei Luo1, Min Liang2, Boyun Shi2, Xinke Zhou2* and Zhixiang Jian3*

Abstract Background: Liver tumor initiating cells (TICs) have self-renewal and differentiate capacities, and largely contribute to tumor initiation, metastasis and drug resistance. MAPK signaling is a critical pathway in many biological processes, while its role in liver TICs hasn’tbeenexplored. Methods: Online-available dataset was used for unbiased screening. Liver TICs were examined CD133 FACS or oncosphere formation. TIC self-renewal was detected by oncosphere formation and tumor initiation assay. LncRNA function was detected by loss of function or gain of function assays. The molecular mechanism of lncRNA was explored by RNA pulldown, RNA immunoprecipitation, ChIP, western blot and double FISH. Results: Here, we examined the expression profiles of MAPK components (MAPKs, MAP2Ks, MAP3Ks, MAP4Ks), and found MAPK6 is most highly expressed in liver cancer samples. Moreover, a divergent lncRNA (long noncoding RNA) of MAPK6, termed lncMAPK6 here, is also overexpressed along with liver tumorigenesis. LncMAPK6 promotes liver tumor propagation and TIC self-renewal through MAPK6. LncMAPK6 interacts with and recruits RNA polymerase II to MAPK6 promoter, and finally activates the transcription of MAPK6. Through MAPK6 transcriptional regulation, lncMAPK6 drives MARK signaling activation. LncMAPK6-MAPK6 pathway can be used for liver TIC targeting. Altogether, lncMAPK6 promotes MARK signaling and the self-renewal of liver TICs through MAPK6 expression. Conclusion: MAPK6 was the most highly expressed MAPK component in liver cancer and liver TICs and lncMAPK6 participated in the transcriptional regulation of MAPK6in cis. This work revealed the importance role of MAPK signaling in liver TIC self-renewal and added a new layer for liver TIC and MAPK6 expression regulation. Keywords: MAPK6, lncMAPK6, Liver TIC, Self-renewal, Tumor targeting

Background sphere formation, side-population, transwell and xeno- Liver cancer is a serious cancer type all over the world. graft [9, 15, 24]. In these assays, sphere formation and Liver cancer bulk contains many kinds of cells, including gradient xenograft formation were widely-accepted for liver tumor initiating cells. Liver tumor initiating cells liver TICs. Some surface markers have been examined to (TICs), also termed liver cancer stem cells (CSCs), are detect and enrich liver TICs, including CD133, CD13, the origin of liver tumorigenesis, metastasis, drug- CD24 and CD90 [11, 21, 38]. However, the biology of resistance and relapse [16]. Liver TICs can self-renewal liver TICs remains elusive. and differentiate into a new tumor [33, 34]. Recently, Several transcription factors participate in the self- several functional assays of liver TICs have been renewal of liver TIC, including Zic2, c-Myc, Oct4, Sox4 developed, including gradient xenograft formation, [8, 41]. The well-known modulators of liver TIC self- renewal are Wnt/β-catenin signaling, Notch signaling, * Correspondence: [email protected]; [email protected] Hedgehog and Yap1 signaling pathways [30, 40, 42, 43]. †Equal contributors MAPK signaling pathway, one of the most important 2Department of Abdominal Oncology, The Fifth Affiliated Hospital of pathways in signaling transduction and an integration Guangzhou Medical University, Guangdong Sheng, China 3Department of General Surgery, Guangdong General Hospital, Guangdong point for multiple biochemical signals, plays a critical Academy of Medical Sciences, Guangdong Sheng, China role in tumorigenesis, immunology regulation, growth Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:105 Page 2 of 11

and development [14, 20, 28, 44]. However, the role of Antibodies and reagents MAPK signaling pathway in liver TICs hasn’t been Anti-β-actin antibody and DAPI were purchased from explored. Here we performed a unbiased screening on Sigma-Aldrich. Anti-MAPK6 and anti-POLR2A the expression profile of MAPK components in liver antibodies were from Proteintech. PE-CD133 was tumorigenesis, and found MAPK6 (mitogen-activated obtained from MiltenyiBiotec. T7 RNA polymerase protein kinase 6) was the most highly expressed MAPK and Biotin RNA Labeling Mix were purchased from component in liver cancer and liver TICs. Roche. The LightShift Chemiluminescent RNA EMSA MAPK6 is a member of the Ser/Thr protein kinase and Chemiluminescent Nucleic Acid Detection kits family and also termed as Erk3. MAPK6 is related to were from Thermo Scientific. Arrhythmogenic right ventricular cardiomyopathy (ARVC) and p70S6K Signaling, and associates with pul- monary immaturity [3, 19]. It is known that MARK6 Oncosphere formation can be phosphorylated at ser-189 and then activate The oncosphere formation assay was performed as MAPKAPK5, which in turn phosphorylates MARK6 described [40]. Briefly speaking, 5000 HCC cells were [2]. By similarity, MAPK6 may promote cell cycle en- cultured in sphere formation medium (N2, B27, trance. As for tumorigenesis, MARP6 participates in 20 ng/ml EGF and 20 ng/ml bFGF, in DMEM/F12) HCC development and non-small-cell lung cancer and seeded into Ultra Low Attachment 6-well plates. (NSCLC) development [5, 6]. While, its role in liver TICs Two weeks later, oncospheres were counted typical is unclear. Here we found MAPK6 is vigorously expressed images were taken. in liver tumor and TICs. MAPK6 deficient liver TICs showed impaired self-renewal, and MAPK6 overexpres- Tumor propagation and initiating assay sion promoted the self-renewal of liver TICs, revealing the 6 critical role of MAPK6 in liver TICs. For tumor propagation, 1 × 10 lncMAPK6 silenced, Long noncoding RNAs (LncRNAs) are defined as overexpressed or control cells were injected into BALB/c nude mice, which were satisficed 1 month later and RNA molecules which are longer than 200 nucleo- 2 ’ tumors were weighted. For tumor-initiation, 10, 1 × 10 , tides in length but can tcodeprotein[4]. Recent 3 4 5 studies defined lncRNAs as critical modulators in 1×10 ,1×10, and 1 × 10 lncMAPK6 silenced cells or various physiological and pathological processes, in- overexpressed cells were injected into 6-week-old BALB/ cluding tumorigenesis [29, 35]. As for tumorigenesis, c nude mice. The ratios of tumor-free mice and tumor lncRNAs are involved in tumor formation, colony TIC ratios were calculated 3 months later. For each formation, metastasis, energy metabolism and so on sample, six mice were used for tumor injection. [17, 32, 37, 39]. LncRNAs can combine with chroma- tin remodeling complexes (SWI/SNF, NURD, NURF, Immunohistochemistry PRC1/2, and so on) and regulate the transcription of Immunohistochemistry (IHC) was performed as described target genes [35]. LncRNAs also interact with signaling [41]. For IHC, Tris/EDTA buffer was used for antigen proteins and change their stability or activity [32, 36]. retrieval. The samples were detected by HRP substrates Here, we found lncMAPK6 and MAPK6 were vigor- and observed with Nikon-EclipseTi microscopy. ously expressed in liver TICs. LncMAPK6 and MAPK6 were involved in the maintenance regulation of liver TICs. LncMAPK6 interacted and recruited RNA pulldown RNA polymerase II to MAPK6 promoter to initiate RNA pulldown assay was performed as described [27]. transcription. Moreover, lncMAPK6-MAPK6 pathway Briefly speaking, biotin-labeled lncMAPK6 transcript serve as a target for liver propagation and TIC was obtained through in vitro transcription, and incu- clearance. baed in sphere lysate. Streptavidin beads were then added for component enrichment, followed by Western blot or silver staining. Methods Primary samples The detailed information of these sample was: #1, early Statistical analysis HCC, 61 years old, female, tumor size, 7.2 × 6.3 × 5.2 mm, For statistical analysis, one-tailed Student’s t tests were non-metastasis. #4, advanced HCC, 74 years old, male, used. P < 0.05 was defined to be statistically significant. tumor size, 7.9 × 5.3 × 4.8 mm, metastasis. #5, advanced Other than denoted, data were shown as means±s.d. HCC, 63 years old, female, tumor size, 6.9 × 6.1 × 4.2 mm, *P <0.05;**P < 0.01; ***P < 0.001. All experiments were non-metastasis. repeated at least three times. Huang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:105 Page 3 of 11

Results cancer samples (Fig. 1a, b). MAPK6 overexpression levels MAPK6 was up-regulated in liver tumor and liver TICs are related to clinical survival (Fig. 1c). To further examine Liver TICs are important for liver tumorigenesis, relapse, the high expression of MAPK6 in liver cancer, we detected metastasis and drug resistance. MAPK signaling pathway MAPK6 expression levels with realtime PCR and Western is a integration point for multiple biochemical signals. blot. MAPK6 was up-regulated along with liver tumori- However, the role of MAPK pathway in liver TICs hasn’t genesis (Fig. 1d, e). Of note, MAPK6 expression was also been explored. Here we performed a unbiased screening related to cancer severity (Fig. 1d, e). for the expression levels of MAPK components in liver We then examined MAPK6 expression pattern in liver cancers using GSE14520 dataset [23, 26]. Of the MAPK TICs. We isolated liver TICs through surface marker components, MAPK6 is most highly expressed in liver CD133, and found MAPK6 was highly expressed in liver

Fig. 1 Overexpression of MAPK6 in liver tumor and TICs. (a) Expression profiles of MAPK components were shown as heatmap. Blue denoted low expression and red denoted high expression. (b) MAPK6 is highly expressed in liver cancers. (c) According to MAPK6 expression, HCC samples were divided into two groups (MAPK6high and MAPK6low)forKaplan–Meier survival analysis. (d) MAPK6 expression were examined in 19 peri-tumor samples, 7 early HCC samples (eHCC) and 12 advanced HCC samples (aHCC), and all the results were normalized to the average of peri-tumor expression. (e) Immunohistochemistry of MAPK6 in peri-tumor, eHCC and aHCC. Representative images and indicated ratios were shown. (f, g)MAPK6 mRNA expression in CD133+ liver TICs (f) or oncospheres (g) was examine with realtime PCR. RNA expression levels were normalized to MAPK6 expression in non-TICs. For b, data were presented as box and whisker plot, boxes represent interquartile ranges (IQR) Huang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:105 Page 4 of 11

TICs (Fig. 1f). We performed sphere formation assay, lncMAPK6 expression in liver TICs (Fig. 2d). We also and also found high expression of MAPK6 in onco- performed sphere formation assay and found spheres (Fig. 1g). increased lncMAPK6 expression in oncospheres (Fig. 2e). Fluorescence in situ hybridization (FISH) also confirmed High expression of lncMAPK6 in liver cancer and liver TICs the high expression of lncMAPK6 in liver TICs (Fig. 2f) We then explored the regulation mechanism of MAPK6 and oncospheres (Fig. 2g). Altogether, lncMAPK6 was expression. Firstly, we observed MAPK6 locus, and highly expressed in liver tumor and TICs. found lncRNA ENST00000561318 (hereafter named lncMAPK6) is a divergent lncRNA of MAPK6 (Fig. 2a). LncMAPK6 activated liver TIC self-renewal Through Northern blot, lncMAPK6 was also highly We also detected the role of lncMAPK6 in liver TIC expressed in HCC samples (Fig. 2b). We then examined regulation. LncMAPK6 depleted cells were generated for lncMAPK6 expression profile through in situ hybridization oncosphere formation assay. Sphere formation was (ISH). As shown in Fig. 2c, lncMAPK6 was highly inhibited once lncMAPK6 was depleted, indicating the expressed in liver cancer. essential role of lncMAPK6 in liver TIC self-renewal We also detected lncMAPK6 expression in liver TICs. (Fig. 3a). We also injected 10, 1 × 102,1×103,1×104 We enriched CD133+ liver TICs, examined lncMAPK6 and 1 × 105 lncMAPK6 depleted cells into BALB/c nude expression with realtime PCR, and found increased mice for tumor initiation, and found impaired tumor

Fig. 2 LncMAPK6 is overexpressed in liver tumor and TICs. (a) MAPK6 locus was shown. There is a divergent lncRNA (ENST00000561318, termed as lncMAPK6 in this research) in near from MAPK6 locus according to UCSC Genome Browser. (b) LncMAPK6 expression levels were detected using Northern blot. 18S rRNA was a loading control. (c) 19 peri-tumor, 7 eHCC and 12 aHCC were used for In situ hybridization (ISH). Representative photos and indicated ratios were shown. (d) LncMAPK6 expression levels in CD133+ liver TICs and CD133− non-TICs were detected by realtime PCR, and non-TIC expression was used for data normalization. (e) Spheres and non-spheres were selected, and lncMAPK6 expression was examined with realtime PCR. (f, g) FISH assay confirmed the high expression of lncMAPK6 in liver TICs (f) and oncospheres (g). Zic2 and CD133 were markers of liver TICs. Scale bars, C, 50 μm; F, 20 μm Huang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:105 Page 5 of 11

Fig. 3 LncMAPK6 was required for liver TIC self-renewal. (a) LncMAPK6 depleted cells were generated through CRISPRi strategy, and sphere formation assay was performed to detect liver TIC self-renewal. LncMAPK6 depletion efficiency was examined by Northern blot (left). Typical photos and the ratios of sphere-initiating cells were shown. (b) 10, 1 × 102,1×103,1×104 and 1 × 105 lncMAPK6 depleted cells were used for 3 months’ tumor formation. Tumor initiating cell ratios were analyzed through extreme limiting dilution analysis (right panels). CI, confidence interval. (c) Transwell assay was performed using lncMAPK6 silenced cells, and representative images and cell numbers were shown. (d, e) LncMAPK6 antisense oligo (ASO) treated cells were utilized for sphere formation (d) and invasion assay (e), and the typical images were shown. (f) LncMAPK6 overexpressed cells were established by CRISPRa strategy, and enhanced sphere formation capacity was found in lncMAPK6 overexpressing cells. (g) 10, 1 × 102,1×103,1×104 and 1 × 105 lncMAPK6 overexpressed cells were used for 3 months’ tumor initiation initiation (Fig. 3b). The ratios of liver TICs were also overexpressed cells for tumor initiation, and found decreased upon lncMAPK6 depletion (Fig. 3b). We also lncMAPK6 overexpression promoted tumor formation found lncMAPK6 was required for the invasion of liver (Fig. 3g). Altogether, lncMAPK6 played a important role TICs (Fig. 3c). We also silenced lncMAPK6 through in the self-renewal of liver TICs. antisense oligo (ASO) and performed sphere formation and transwell assays. The results confirmed that LncMAPK6 drove MAPK6 expression lncMAPK6 was required for the self-renewal and inva- Many lncRNAs participate in the transcription of nearby sion of liver TICs (Fig. 3d, e). genes, thus we detected whether lncMAPK6 drove the To further confirm the role of lncMAPK6 in liver TICs, expression of MAPK6. We examined MAPK6 expression we then overexpressed lncMAPK6 through CRISPRa levels with lncMAPK6 depleted cells and found de- strategy, and found lncMAPK6 overexpression promoted creased MAPK6 expression upon lncMAPK6 knock- the self-renewal of liver TICs (Fig. 3f). Then we injected down (Fig. 4a). Western blot also found that lncMAPK6 10, 1 × 102,1×103,1×104 and 1 × 105 lncMAPK6 depletion largely impaired MAPK6 expression (Fig. 4b). Huang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:105 Page 6 of 11

Fig. 4 (See legend on next page.) Huang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:105 Page 7 of 11

(See figure on previous page.) Fig. 4 LncMAPK6 drove MAPK6 expression. (a) MAPK6 expression in lncMAPK6 depleted cells was detected using three primary samples. (b, c) LncMAPK6 depleted (b) and overexpressed (c) cells were used for MAPK6 examined with immunobloting. CRISPRi-lnc, CRISPRi-lncMAPK6; CRISPRa-lnc, CRISPRa- lncMAPK6. β-actin was a loading control. (d) MAPK6 knockout cells were established and sphere formation assay was performed. Typical images and indicated ratios were shown in middle and right panels, respectively. (e) 10, 1 × 102,1×103,1×104 and 1 × 105 MAPK6 knockout cells were used for tumor formation. (f, g) LncMAPK6 was silenced (f) or overexpressed (g) in MAPK6 knockout cells and lncMAPK6 didn’t participate in liver TIC self-renewal upon MAPK6 was deleted. (h, i) MAPK6 knockout cells were used for lncMAPK6 knockdown (h) or overexpression (i), followed by transwell assay

On the contrary, lncMAPK6 overexpression promoted the knockout cells contained decreased ratios of liver TICs expression of MAPK6 (Fig. 4c). Altogether, lncMAPK6 (Fig. 4e). Altogether, MAPK6 was essential in liver TIC drove the expression of MAPK6. self-renewal. Next we detected the role of MAPK6 in liver TICs. We also detected the importance of MAPK6 for MAPK6 knockout cells were constructed and showed im- lncMAPK6 function. We silenced lncMAPK6 expression paired sphere formation capacity, indicating that MAPK6 in MAPK6 knockout cells, followed by sphere formation. was necessary for liver TIC self-renewal (Fig. 4d). We also Interestingly, lncMAPK6 had no effect on liver TIC self- performed tumor initiation with gradient MAPK6 knock- renewal upon MAPK6 knockout, revealing the critical out cells, and found MAPK6 knockout largely impaired function of MAPK6 in lncMAPK6 (Fig. 4f). Similarly, we tumor initiation capacity (Fig. 4e). Meanwhile, MAPK6 overexpressed lncMAPK6 in MAPK6 knockout cells and

Fig. 5 LncMAPK6 interacted with RNA polymerase II. (a) LncMAPK6 was transcribed in vitro for RNA pulldown, and the denoted band in lncMAPK6 sample was identified as POLR2A. (b) The binding of lncMAPK6 and POLR2A was examined by Western blot. (c) LncMAPK6 truncates were generated (left panels), and incubated with sphere lysates. The interaction between lncMAPK6 truncates and POLR2A was confirmed (right panels). (d) RNA EMSA was performed for the combination between lncMAPK6 and POLR2A. The second truncate of lncMAPK6 was used for RNA EMSA. (e) Oncospheres derived from clinical samples were used for RNA immunoprecipitation (RIP) assay, and enrichment of lncMAPK6 were detected with realtime PCR. GAPDH was a control. (f) The co-localization of lncMAPK6 and POLR2A was confirmed by double FISH assay. Scale bars, 10 μm. (g, h) LncMAPK6 depleted (g) and overexpressed (h) cells were crushed for POLR2A ChIP, followed by detection for MAPK6 promoter enrichment with realtime PCR Huang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:105 Page 8 of 11

found impaired role of lncMAPK6 (Fig. 4g). Beside sphere also validated by Western blot (Fig. 5b). LncMAPK6 formation assay, tumor invasion capacity was also exam- truncates were also constructed for RNA pulldown. ined. LncMAPK6 had no effect on tumor invasion if The second region of lncMAPK6 (#2) was required MAPK6 was knockout, further confirming the importance for binding with POLR2A (Fig. 5c). EMSA experiment of MAPK6 in lncMAPK6 signaling (Fig. 4h, i). Altogether, also confirmed the combination of lncMAPK6 and lncMAPK6 drove liver TIC self-renewal by MAPK6. POLR2A (Fig. 5e). We also examined the physiological interaction of POLR2A and lncMAPK6 with LncMAPK6 recruits RNA polymerase II to MAPK6 promoter RNA immunoprecipitation (Fig. 5e). Double fluores- To investigate the molecular mechanisms of lncMAPK6 in cence in situ hybridization (double-FISH) was also MAPK6 expression and subsequent liver TIC self-renewal, confirmed the co-localization of lncMAPK6 and RNA pulldown assay was performed. Mass spectrum was POLR2A (Fig. 5f). used for protein identification and the interaction between POLR2A, a component of RNA polymerase II, plays a lncMAPK6 and POLR2A was found (Fig. 5a), which was fundamental role in transcriptional initiation. Thus we

Fig. 6 LncMAPK6-MAPK6 pathway can be targeted for liver TIC clearance. (a, b)1×106 lncMAPK6 silenced (ASO, A) or overexpressed (oeLnc, b) were used for 1 month tumor propagation. 6 BALB/c nude mice were used for each sample. (c)1×106 indicated cells were used for tumor propagation and tumor weights were examined one month later. (d) The indicated tumors were used for MAPK6 immunohistochemistry. (e, f) The indicated tumors were stained with Ki67 (e) and CD133 (f) antibodies for FACS detection. Gating strategy and indicated ratios were shown Huang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:105 Page 9 of 11

explored the role of lncMAPK6-POLR2A interaction MAPK6 through transcriptional activation. There are sev- in MAPK6 transcription. We found POLR2A com- eral lncRNAs involved in MAPK modulation. MALAT1 bined with MAPK6 promoter in liver TICs, while, drove the proliferation and metastasis of gallbladder can- their binding was diminished upon lncMAPK6 was cer cells through MAPK signaling [1]. DBH-AS1 and depleted (Fig. 5g). On the contrary, lncMAPK6 overex- URHC are required for the proliferation and survival of pression largely promoted the binding of POLR2A and hepatocellular carcinoma by MAPK activation [13, 18]. MAPK6 promoter (Fig. 5h). Altogether, lncMAPK6 Here we found lncMAPK6 promoted MAPK6 expression recruited RNA polymerase II to the promoter of MAPK6 to drive liver TIC self-renewal, revealing the role of to drive its expression. MAPK signaling in liver TIC self-renewal and adding a new layer for MAPK6 transcriptional regulation. LncMAPK6-MAPK6 served as a target for liver TIC clearance As we know, It is impossible that a lncRNA only target Finally, we detected the TIC targeting role of lncMAPK6- one gene. However, we found lncMAPK6 had impaired MAPK6. We examined the tumor propagation capacity of role upon MAPK6 was deleted, indicating lncMAPK6 lncMAPK6 depleted cells and lncMAPK6 overexpressed exerted its role mainly through MAPK6. We think several cells, and found lncMAPK6 played an important role in reasons can explain this result. 1. Some lncRNAs may lo- liver tumor propagation (Fig. 6a, b). We also established cate at particular subcellular position, and the location MAPK6 promoter deleted cells, followed by tumor propa- limited the number of their target genes. 2. lncRNA tar- gation. MAPK6 promoter or MAPK6 knockout cells also geted some other genes, but these genes weren’trequired showed impaired propagation (Fig. 6c). We performed for the self-renewal of liver TICs; 3. Some lncRNAs partic- immunohistochemistry assay, and the results showed ipated in the transcriptional regulation through lncRNA impaired expression of MAPK6 in lncMAPK6 knockout transcription process but not lncRNAs themself [31]. The or MAPK6 promoter depleted tumors, validating the role process of lncRNA can activate the transcription of their of lncMAPK6 in MAPK6 expression (Fig. 6d). We nearby genes. Meanwhile, our results indicated that obtained tumor cells and found lncMAPK6-MAPK6 MAPK6 participated in the self-renewal of liver TICs, but pathway was required for the proliferation of tumor cells the molecular mechanism of MAPK6 function is still un- (Fig. 6e). Meanwhile, lncMAPK6-MAPK6 inhibition also clear, which need further investigation. Altogether, induced a decreased numbers of liver TICs, indicating that lncMAPK6 drove the self-renewal of liver TICs through lncMAPK6-MAPK6 can be used for liver TIC clearance MAPK6 expression. LncMAPK6-MAPK6 pathway can be (Fig. 6f). Taken together, lncMAPK6-MAPK6 blockade used for liver TIC targeting. inhibited the self-renewal and maintenance of liver TICs. Conclusion Discussion MAPK6 was the most highly expressed MAPK compo- Wnt/β-catenin, Notch and Hedgehog signaling pathways nent in liver cancer and liver TICs and lncMAPK6 par- are well-explored pathways in liver TIC self-renewal. All ticipated in the transcriptional regulation of MAPK6in these signaling pathways are finely regulated. Wnt/β-ca- cis. This work revealed the importance role of MAPK tenin activation is largely modulated by β-catenin stabil- signaling in liver TIC self-renewal and added a new layer ity or activity [7, 10, 22]. Notch activation is regulated by for liver TIC and MAPK6 expression regulation. Notch cleavage and nuclear translocation [40]. Here we found MAPK6, a component of MAPK signaling path- Acknowledgements ’ The authors thank Yueming He (Department of Abdominal Oncology, The Fifth way, is involved in the self-renewal of liver TICs. What s Affiliated Hospital of Guangzhou Medical University) for experimental supports. more, MAPK6 is also precisely regulated by lncMAPK6. LncMAPK6 located onto the MAPK6 promoter and re- Funding MAPK6 This work was supported by science and technology projects of Guangdong cruited RNA polymerase II to promoter, which province (2017A020211031, 2016ZC0145), science and technology projects of further activate the transcription activation of MAPK6. Guangzhou Medical University (201624), the National Natural Science LncRNAs play important roles in many biological pro- Foundation of China (81673206) and science and technology projects of Guangzhou city (201508020262), Medical Scientific Research Foundation of cesses [25]. Here we found a lncRNA drove the self- Guangdong province (A2015243). renewal of liver TICs through MAPK6. Interestingly, lncMAPK6 is a divergent lncRNA of MAPK6. Divergent Availability of data and materials lncRNAs are transcribed in the opposite direction from All data and materials can be provided upon request (email [email protected] or [email protected]). nearby genes [12]. Divergent lncRNAs, often modulate their nearby genes in cis, are positively-related to their Authors’ contributions nearby genes [12]. As a divergent lncRNA of MAPK6, GH and HJ performed experiments, analyzed data and wrote the paper; YH, LY, WX, YL, ML and BS performed some experiments and analyzed data; lncMAPK6 was co-expressed with MAPK6 in liver XZ and ZJ initiated the study, designed experiments and wrote the paper. tumor and TICs. LncMAPK6 regulated the expression of All authors read and approved the final version of the manuscript. Huang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:105 Page 10 of 11

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