FOXK2 Downregulation Suppresses EMT in Hepatocellular Carcinoma

Open Medicine 2020; 15: 702–708

Research Article

Jian Kong#, Qingyun Zhang#, Xuefeng Liang, Wenbing Sun* FOXK2 downregulation suppresses EMT in hepatocellular carcinoma

https://doi.org/10.1515/med-2020-0129 Keywords: hepatocellular carcinoma, FOXK2, epithe- received December 22, 2019; accepted March 31, 2020 lial–mesenchymal transition, Akt Abstract: Forkhead box K2 (FOXK2) was first identified as an NFAT-like interleukin-binding factor. FOXK2 has been reported to act as either oncogene or tumor suppressor. However, functional and regulating mechan- 1 Introduction isms of FOXK2 in epithelial–mesenchymal transition ( ) (EMT) in hepatocellular carcinoma (HCC) remain un- Hepatocellular carcinoma HCC is listed as the sixth clear. An FOXK2-specific siRNA was employed to most common neoplasm and the third leading cause of decrease the endogenous expression of FOXK2. MTT cancer death, which has been recognized as a major [ ] assay, colony formation and transwell assay were used cause of death among cirrhotic patients 1 . Surgical to evaluate proliferation, migration and invasion of resection, liver transplantation and radiofrequency [ ] Hep3B and HCCLM3 cells, respectively. The protein ablation are considered potentially curative for HCC 2 . expression associated with EMT and Akt signaling However, most HCC patients are diagnosed at advanced pathways was evaluated using western blot. FOXK2 stages when surgical treatments are unsuitable. Only downregulation could inhibit cell proliferation and sorafenib and regorafenib are proven to prolong survival [ – ] colony formation and suppress migration and invasion in HCC 3 8 . Therefore, development of new targeted in Hep3B and HCCLM3 cells. The expression of E- therapy for HCC treatment is urgent. ( ) cadherin was significantly upregulated, and the expres- The structure of Forkhead box FOX proteins contains fi sion of snail and p-Akt was significantly downregulated conserved DNA binding domain, which is de ned as the - - in siFOXK2-transfected cells compared with control cells. forkhead winged helix turn helix. FOX proteins participate - SF1670 induced the expression of p-Akt and snail and in various biological processes, which include metabo ff - suppressed the expression of E-cadherin in Hep3B and lism, development, di erentiation, apoptosis, prolifera [ ] HCCLM3 cells. SF1670 promoted the invasion and colony tion, migration, invasion and longevity 9 . Forkhead box ( ) formation of Hep3B and HCCLM3 cells. SF1670 partly K2 FOXK2 , also regarded as an interleukin enhancer fi inhibited the effect of FOXK2 suppression on Hep3B and binding factor, was initially con rmed as a nuclear factor - - HCCLM3 cells. In conclusion, this study revealed that of an activated T cell like interleukin binding factor. In FOXK2 downregulation suppressed the EMT in HCC human tumors, FOXK2 has been reported to act as either [ ] partly through inhibition of the Akt signaling pathway. oncogene or tumor suppressor 10 .Inglioma,FOXK2 could inhibit the cell multiplication and motility and predict a favorable prognosis [11].Inclear-cell renal cell  carcinoma, FOXK2 expression was downregulated in # Jian Kong and Qingyun Zhang contributed equally to this work. tumor tissue, which suppressed the capabilities of proliferation and motility and promoted apoptosis via  [ ] * Corresponding author: Wenbing Sun, Department of suppression of epidermal growth factor receptor 12 .In Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical breast cancer, FOXK2 could regress various genes con- University, Beijing 100043, China, e-mail: [email protected] taining HIF1β and EZH2, which further inhibited the Jian Kong: Department of Hepatobiliary Surgery, Beijing Chaoyang hypoxia-induced response and tumor carcinogenesis [13]. Hospital, Capital Medical University, Beijing 100043, China In HCC, FOXK2 promoted cell growth and indicated ffi Qingyun Zhang: Department of General Surgery, A liated Hospital [ ] α- of Chengde Medical University, Hebei 067000, China unfavorable prognosis 14 .InER positive breast cancer, Xuefeng Liang: Blood center of Shandong Province, Shandong FOXK2 could inhibit tumor growth via decreasing the ERα 250000, China stability through BRCA1/BARD1-associated mechanisms

[15]. In colorectal cancer, Sox9 could activate FOXK2 and was added to each well at a final concentration of 5 mg/ further promote the tumor cell multiplication [16].FOXK2 mL and incubated at 37°C for 4 h. Dimethyl sulfoxide could translocate DVL into the nucleus and further (150 µL) was used to dissolve the formazan crystals. The activate the Wnt/β-catenin signaling pathway [17].In absorbance was detected at 570 nm using a microplate breast cancer, FOXK2 could also mediate epirubicin and reader (Bio-Rad Laboratories, USA). paclitaxel resistance via FOXO3a [18]. However, the concrete mechanism of FOXK2 in HCC is still unclear. High rates of tumor recurrence and metastasis lead to poor prognosis in patients with advanced HCC [19]. 2.3 Colony formation assay Metastasis is a complicated biological process, which is involved in a poly-stage cascade of genetic and Briefly, Hep3B or HCCLM3 cells with or without FOXK2 epigenetic events [20]. Epithelial–mesenchymal transi- knockdown were trypsinized, and 1 × 103 cells/well were tion (EMT) plays a crucial role in tumor metastasis. added into six-well plates. The cells were continually During the process of EMT, polarized epithelial cells cultured at 37°C for 14 days. Wells were stained with transform into fibroblast-like mesenchymal cells, and 0.1% crystal violet (Solarbio, China) for 20 min before the cells become more motile and invasive and more counting colonies under a dissecting microscope. susceptible to metastasis [21–24]. Transforming growth factor-β (TGF-β), which is increasing in cirrhosis and late-stage HCC, could induce EMT in HCC [25]. FOXK2 could inhibit the EMT of non-small cell lung cancer 2.4 Migration and invasion assays (NSCLC)[26]. Whether FOXK2 is involved in the process of EMT in HCC remains uncertain. After FOXK2 knockdown, Hep3B or HCCLM3 cells were In the present study, we evaluated the biological collected, and 2 × 104 cells or 5 × 104 cells in the medium functions of FOXK2 using HCC cell line and the effect of were added to the upper chamber of transwell plates FOXK2 knockdown on EMT, the malignant phenotype (Corning, USA) with or without BD Matrigel (BD and associated signaling pathways. We demonstrated Biosciences, USA) matrix coating. Medium containing that FOXK2 downregulation suppressed the EMT in HCC 10% FBS was added into the lower chambers. After 24 h cell lines through inhibition of the Akt pathway. culture, the non-migrating or non-invading cells in the upper chamber were gently removed, and the migrating or invading cells were stained with 0.1% crystal violet (Solarbio, China) for 20 min, imaged and counted under 2 Materials and methods a microscope.

2.1 Cell lines and cell culture 2.5 Gene transfection Hep3B and HCCLM3 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, Hep3B or HCCLM3 cells were cultured in six-well plates China). The cells were cultured in Dulbecco’s modiﬁed at a density of 5 × 105 cells/well. The gene transfection Eagle’s medium (DMEM; Gibco, USA) supplemented with assay was performed with the following constructs using 10% fetal bovine serum (FBS; Gibco, USA) and incubated Lipofectamine 2000 (Invitrogen, USA) according to the in 5% CO2 at 37°C. manufacturer’s instructions. FOXK2 siRNA sequences were as follows: 5′-GCGAGUUCGAGUAUCUGAUTT-3′ (sense) and 5′-AUCAGAUACUCGAACUCGCTT-3′ (antisense). Negative control siRNA sequences were as 2.2 Cell proliferation assay follows: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense). Cells Cell proliferation was assessed using the MTT method. were cultured with siRNA in DMEM for 6 h, then the Brieﬂy, cells were seeded at 3 × 103 cells/well into a 96- medium was replaced with DMEM containing 10% FBS. The well plate and were cultured for 24, 48 or 72 h. In the end cells were incubated for 48 h and collected for next of the incubation, MTT (Sigma-Aldrich, USA) solution experiments. The concentration of siRNA was 100 nmol/L. 704  Jian Kong et al.

Figure 1: FOXK2 downregulation inhibits cell growth and colony formation in Hep3B and HCCLM3 cells. The expression of FOXK2 was knocked down in Hep3B or HCCLM3 cells to define the biological effect of FOXK2. (a) The efficiency of knockdown of FOXK2 was confirmed by western blot. (b) FOXK2 knockdown dramatically inhibited cell proliferation of Hep3B or HCCLM3 cells at 48 and 72 h. (c) FOXK2 knockdown also dramatically inhibited the colony formation of Hep3B or HCCLM3 cells. These data are representative of at least three independent experiments. ns: no significance; **P < 0.01 and ***P < 0.001.

2.6 Western blot analysis 3 Results

Total proteins were extracted using RIPA containing 3.1 FOXK2 downregulation inhibits cell protease and phosphatase inhibitors. Protein concentra- tions were measured by the bicinchoninic acid method. growth and colony formation in Hep3B The protein samples (50 µg) were separated by SDS- or HCCLM3 cells PAGE and transferred onto nitrocellulose membranes ( ) EMD Millipore, USA . Immunoblotting was performed To determine the biological effect of FOXK2 on HCC, the β- ( ) - with antibodies against actin 1:2,000 ,Ecadherin FOXK2expressionwasknockeddowninHep3BorHCCLM3 ( ) - ( ) - ( ) ( ) 1:1,000 ,pAkt 1:500 ,tAkt 1:1,000 , snail 1:500 cells. The efficiency of FOXK2 knockdown was confirmed by ( ) ( Abcam, Cambridge, MA, USA and FOXK2 1:500; CST, western blot (Figure 1a). We analyzed the influence of FOXK2 ) Boston, MA, USA . Then, the blot was examined with on cell proliferation. FOXK2 knockdown dramatically inhib- - ( ) - HRP labeled secondary antibody 1:4,000 . The visuali ited cell proliferation of Hep3B or HCCLM3 cells at 48 and zation of protein bands was detected using the ECL 72 h (Figure 1b). Meanwhile, FOXK2 knockdown also ( ) detection system Millipore, USA according to the dramatically inhibited the colony formation of Hep3B or ’ manufacturer s instructions. HCCLM3 cells (Figure 1c). Similar results were also found using another FOXK2 siRNA (Supplementary Figure 1).

2.7 Statistical analysis 3.2 FOXK2 knockdown suppresses the All data are presented as mean ± SD. GraphPad Prism8 migration and invasion of Hep3B or (GraphPad Software, La Jolla, CA, USA) was used for HCCLM3 cells statistical analysis. Comparison between two or multiple groups was performed using Student’s t-test or one-way We also explored the eﬀect of FOXK2 on the migration analysis of variation. P < 0.05 was considered statisti- and invasion of Hep3B or HCCLM3 cells. Migration assay cally signiﬁcant. suggested that migrated cell numbers in siFOXK2- FOXK2 downregulation suppresses EMT in HCC  705

Figure 2: FOXK2 knockdown suppresses the migration and invasion of Hep3B or HCCLM3 cells. We explored the role of FOXK2 in the migration and invasion of Hep3B or HCCLM3 cells. (a and b) Migration assay demonstrated that cell motility was significantly decreased in siFOXK2-transfected cells compared with control cells. (c and d) The matrigel invasion assay showed that invasiveness was significantly suppressed in siFOXK2-transfected cells. These data are representative of at least three independent experiments. ***P < 0.001. transfected cells were significantly less than that of the which is a PTEN-specific inhibitor. SF1670 increased the control group in Hep3B and HCCLM3 cells, which expression of p-Akt and snail and decreased the demonstrated that cell motility was significantly de- expression of E-cadherin (Figure 3c and d). creased after FOXK2 knockdown (Figure 2a and b). We also observed the effect of SF1670 on FOXK2 Similarly, the matrigel invasion assay demonstrated that regulating the biological changes in Hep3B or HCCLM3 invasiveness was significantly suppressed in siFOXK2- cells. SF1670 promoted the invasion and colony forma- transfected cells (Figure 2c and d). tion of HCC cells and partly inhibited the effect of FOXK2 inhibition on HCC cells (Figure 4).

3.3 FOXK2 may regulate the EMT in Hep3B or HCCLM3 cells through Akt signaling 4 Discussion

To verify the associated mechanism of FOXK2 involved FOXK2 has been reported to act as either oncogene or in the biological changes in Hep3B or HCCLM3 cells, we tumor suppressor; however, the effect of FOXK2 on the also observed the role of FOXK2 in EMT markers and EMT has not yet been demonstrated in HCC cells. In the related signals. E-cadherin expression was significantly current research, we demonstrated that FOXK2 knock- upregulated, and snail and p-Akt expression was down inhibited the proliferation, colony formation, significantly downregulated in siFOXK2-transfected cells migration and invasion of HCC cells. Furthermore, we compared with control cells (Figure 3a and b).To describe a possible mechanism by which FOXK2 down- confirm whether the Akt signaling pathway was involved regulation inhibited EMT. In particular, FOXK2 down- in the process of FOXK2 regulating the EMT in Hep3B or regulation suppressed the EMT of HCC cells partly via HCCLM3 cells, SF1670 was used to activate PI3K/Akt, inhibition of the Akt pathway. 706  Jian Kong et al.

Figure 3: FOXK2 may regulate the EMT in Hep3B or HCCLM3 cells through the Akt signaling pathway. We analyzed the influence of FOXK2 on EMT markers and related signals. (a and b) E-cadherin expression was significantly upregulated and snail and p-Akt expression was significantly downregulated in siFOXK2-transfected cells compared with control cells. (c and d) To confirm whether the Akt pathway participated in the process of FOXK2 regulating the EMT in Hep3B cells, SF1670 was used to activate PI3K/Akt, which is a PTEN-specific inhibitor. SF1670 upregulated the expression of p-Akt and snail and downregulated the expression of E-cadherin. These data are representative of three independent experiments. *P < 0.05 and **P < 0.01.

FOXK2 functions as oncogene in HCC [27] and colorectal proteases, which promoted EMT-associated proteins. TGF- cancer or tumor suppressor in NSCLC, glioma, breast β1isakeyfactorintheprocessofEMT,andE-cadherin on cancer and renal cell carcinoma [12,28]. FOXK2 also the cell surface is cleaved, which disrupts E-cadherin- regulated the sensitivity of breast cancer to epirubicin and mediated cell–cell adhesion and results in EMT [29]. Snail paclitaxel. FOXK2 could suppress cell proliferation and acts as a crucial transcription factor that plays a role as a invasion in clear-cell renal cell carcinoma and breast suppresser of E-cadherin expression and an inducer of cancer and inhibit tumor growth in breast cancer. EMT in diﬀerent kinds of cancer [30].Previousresearch However, FOXK2 was obviously increased in HCC and studies have revealed that the FOXK2 suppressed EMT in associated with tumor size, TNM stage and tumor vascular NSCLC and glioma via suppression of various pivotal invasion, and FOXK2 upregulation in HCC cells could lead target genes [11,26]. In this study, we showed that FOXK2 to increased cell proliferation and migration [27].Our knockdown could increase the E-cadherin expression and results suggested that FOXK2 also functioned as oncogene decrease the Snail expression, which suggested that and loss of FOXK2 inhibited cell proliferation, migration FOXK2 could promote EMT in HCC cells. and invasion in HCC. FOXK2 might be involved in HCC FOXK2 could prevent tumor progression via the p53 carcinogenesis and function as a tumor promoter through pathway, hypoxia pathway or β-catenin signaling suppression of cell proliferation and migration. pathway [13,17]. Furthermore, FOXK2 could also cause EMT facilitates cancer cell invasion and results in oncogenic activity in HCC through the PI3K/Akt sig- tumor metastasis. EMT could be activated by extracellular naling pathway. The PI3K/Akt signaling pathway has FOXK2 downregulation suppresses EMT in HCC  707

Figure 4: Akt activation blocked the effect of FOKK2 inhibition on HCC cells. FOXK2 knockdown dramatically inhibited the invasion (a and b) and colony formation (c) of Hep3B or HCCLM3 cells and SF1670 partly inhibited the effect of FOXK2 suppression on HCC cells. These data are representative of at least three independent experiments. ***P < 0.001. been reported to be involved in the EMT of HCC [31–35]. References The PI3K/Akt signaling pathway might play an impor- [ ] tant role in the FOXK2 regulating EMT in HCC. This study 1 Forner A, Reig M, Bruix J. Hepatocellular carcinoma. Lancet. 2018;391(10127):1301–14. showed that FOXK2 knockdown could decrease the [2] Daher S, Massarwa M, Benson AA, Khoury T. Current and expression of p-Akt, and the activation of PI3K/Akt future treatment of hepatocellular carcinoma: an updated could reverse the process of EMT, which suggested that comprehensive review. J Clin Transl Hepatol. 2018;6(1):69–78. the PI3K/Akt signaling pathway played a crucial role in [3] Raoul JL, Kudo M, Finn RS, Edeline J, Reig M, Galle PR. - the FOXK2 regulating EMT in HCC. Systemic therapy for intermediate and advanced hepatocel lular carcinoma: sorafenib and beyond. Cancer Treat Rev. In conclusion, this work showed that FOXK2 down- 2018;68:16–24. regulation suppressed the EMT progress partly through [4] Bruix J, Qin S, Merle P, Granito A, Huang YH, Bodoky G, et al. inhibition of the PI3K/Akt pathway in HCC cells. These Regorafenib for patients with hepatocellular carcinoma who findings have important implications for understanding progressed on sorafenib treatment (RESORCE): a randomised, the mechanisms and may provide a new therapeutic double-blind, placebo-controlled, phase 3 trial. Lancet. ( ) – target for HCC treatment. 2017;389 10064 :56 66. [5] Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, Kim JS, et al. Efficacy and safety of sorafenib in patients in the Asia-Pacific Acknowledgments: This work was supported by grants region with advanced hepatocellular carcinoma: a phase III from The National Natural Science Foundation of China randomised, double-blind, placebo-controlled trial. Lancet (81502650 and 81572957), Grant QML20190306 from the Oncol. 2009;10(1):25–34. [ ] Beijing Hospitals Authority Youth Program and Grant 2019 6 Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl from the Capital Medical University Incubating Program. J Med. 2008;359(4):378–90. [7] Bouattour M, Mehta N, He AR, Cohen EI, Nault JC. Systemic Conflict of interest: The authors have no conflict of treatment for advanced hepatocellular carcinoma. Liv Cancer. interests. 2019;8(5):341–58. 708  Jian Kong et al.

[8] Finn RS, Merle P, Granito A, Huang YH, Bodoky G, Pracht M, [23] Santamaria PG, Moreno-Bueno G, Portillo F, Cano A. EMT: et al. Outcomes of sequential treatment with sorafenib present and future in clinical oncology. Mol Oncol. followed by regorafenib for HCC: additional analyses from the 2017;11(7):718–38. phase III RESORCE trial. J Hepatol. 2018;69(2):353–8. [24] Goossens S, Vandamme N, Van Vlierberghe P, Berx G. EMT [9] Lam EW, Brosens JJ, Gomes AR, Koo CY. Forkhead box transcription factors in cancer development re-evaluated: proteins: tuning forks for transcriptional harmony. Nature beyond EMT and MET. Biochim Biophys Acta. reviews. Cancer. 2013;13(7):482–95. 2017;1868(2):584–91. [10] Nestal de Moraes G, Carneiro LDT, Maia RC, Lam EW, [25] Giannelli G, Koudelkova P, Dituri F, Mikulits W. Role of Sharrocks AD. FOXK2 transcription factor and its emerging epithelial to mesenchymal transition in hepatocellular carci- roles in cancer. Cancers. 2019;11(3):393. noma. J Hepatol. 2016;65(4):798–808. [11] Wang B, Zhang X, Wang W, Zhu Z, Tang F, Wang D, et al. [26] Chen S, Jiang S, Hu F, Xu Y, Wang T, Mei Q. Foxk2 inhibits non- Forkhead box K2 inhibits the proliferation, migration, and small cell lung cancer epithelial-mesenchymal transition and invasion of human glioma cells and predicts a favorable proliferation through the repression of diﬀerent key target prognosis. OncoTargets Ther. 2018;11:1067–75. genes. Oncol Rep. 2017;37(4):2335–47. [12] Zhang F, Ma X, Li H, Zhang Y, Li X, Chen L, et al. FOXK2 [27] Khemlina G, Ikeda S, Kurzrock R. The biology of hepatocellular suppresses the malignant phenotype and induces apoptosis carcinoma: implications for genomic and immune therapies. through inhibition of EGFR in clear-cell renal cell carcinoma. Mol Cancer. 2017;16(1):149. Int J Cancer. 2018;142(12):2543–57. [28] Jia Z, Wan F, Zhu Y, Shi G, Zhang H, Dai B, et al. Forkhead-box [13] Shan L, Zhou X, Liu X, Wang Y, Su D, Hou Y, et al. FOXK2 elicits series expression network is associated with outcome of massive transcription repression and suppresses the hypoxic clear-cell renal cell carcinoma. Oncol Lett. response and breast cancer carcinogenesis. Cancer Cell. 2018;15(6):8669–80. 2016;30(5):708–22. [29] Dongre A, Weinberg RA. New insights into the mechanisms of [14] Lin MF, Yang YF, Peng ZP, Zhang MF, Liang JY, Chen W, et al. epithelial-mesenchymal transition and implications for FOXK2, regulted by miR-1271-5p, promotes cell growth and cancer. Nat Rev Mol Cell Biol. 2019;20(2):69–84. indicates unfavorable prognosis in hepatocellular carcinoma. [30] Yu H, Shen Y, Hong J, Xia Q, Zhou F, Liu X. The contribution of Int J Biochem Cell Biol. 2017;88:155–61. TGF-beta in epithelial-mesenchymal transition (EMT): down- [15] Liu Y, Ao X, Jia Z, Bai XY, Xu Z, Hu G, et al. FOXK2 transcription regulation of E-cadherin via snail. Neoplasma. factor suppresses ERalpha-positive breast cancer cell growth 2015;62(1):1–15. through down-regulating the stability of ERalpha via me- [31] Hu B, Ding GY, Fu PY, Zhu XD, Ji Y, Shi GM, et al. NOD-like chanism involving BRCA1/BARD1. Sci Rep. 2015;5:8796. receptor X1 functions as a tumor suppressor by inhibiting [16] Qian Y, Xia S, Feng Z. Sox9 mediated transcriptional activation epithelial-mesenchymal transition and inducing aging in of FOXK2 is critical for colorectal cancer cells proliferation. hepatocellular carcinoma cells. J Hematol Oncol. Biochem Biophys Res Commun. 2017;483(1):475–81. 2018;11(1):28. [17] Wang W, Li X, Lee M, Jun S, Aziz KE, Feng L, et al. FOXKs [32] Huang JL, Cao SW, Ou QS, Yang B, Zheng SH, Tang J, et al. The promote Wnt/beta-catenin signaling by translocating DVL into long non-coding RNA PTTG3P promotes cell growth and the nucleus. Dev Cell. 2015;32(6):707–18. metastasis via up-regulating PTTG1 and activating PI3K/AKT [18] Nestal de Moraes G, Khongkow P, Gong C, Yao S, Gomes AR, signaling in hepatocellular carcinoma. Mol Cancer. Ji Z, et al. Forkhead box K2 modulates epirubicin and 2018;17(1):93. paclitaxel sensitivity through FOXO3a in breast cancer. [33] Jiang H, Zhou Z, Jin S, Xu K, Zhang H, Xu J, et al. PRMT9 Oncogenesis. 2015;4:e167. promotes hepatocellular carcinoma invasion and metastasis [19] Clark T, Maximin S, Meier J, Pokharel S, Bhargava P. via activating PI3K/Akt/GSK-3beta/Snail signaling. Cancer Hepatocellular carcinoma: review of epidemiology, screening, Sci. 2018;109(5):1414–27. imaging diagnosis, response assessment, and treatment. Curr [34] Wang R, Yu Z, Chen F, Xu H, Shen S, Chen W, et al. miR-300 Probl Diagn Radiol. 2015;44(6):479–86. regulates the epithelial-mesenchymal transition and invasion [20] Stuelten CH, Parent CA, Montell DJ. Cell motility in cancer of hepatocellular carcinoma by targeting the FAK/PI3K/AKT invasion and metastasis: insights from simple model organ- signaling pathway. Biomed Pharmacother. 2018;103: isms. Nat Rev Cancer. 2018;18(5):296–312. 1632–42. [21] Brabletz T, Kalluri R, Nieto MA, Weinberg RA. EMT in cancer. [35] Xu Q, Liu X, Liu Z, Zhou Z, Wang Y, Tu J, et al. MicroRNA- Nat Rev Cancer. 2018;18(2):128–34. 1296 inhibits metastasis and epithelial-mesenchymal [22] Singh M, Yelle N, Venugopal C, Singh SK. EMT: mechanisms transition of hepatocellular carcinoma by targeting and therapeutic implications. Pharmacol Ther. SRPK1-mediated PI3K/AKT pathway. Mol Cancer. 2018;182:80–94. 2017;16(1):103.