International Journal of

Molecular Sciences Article

Downregulation of FOXP1 Inhibits Cell Proliferation in Hepatocellular Carcinoma by Inducing G1/S Phase Cell Cycle Arrest Xin Wang 1,2 , Ji Sun 1,2 , Meiling Cui 2 , Fangyu Zhao 2 , Chao Ge 2 , Taoyang Chen 3 , Ming Yao 2 and Jinjun Li 2, * 1 2

3

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Shanghai Medical College, Fudan University, Shanghai 200032, China; [email protected] (X.W.); [email protected] (J.S.) State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, 25/Ln 2200, Xietu Road, Shanghai 200032, China; [email protected] (M.C.); [email protected] (F.Z.); [email protected] (C.G.); [email protected] (M.Y.) Pathological Section, Qidong Liver Cancer Institute, Qidong 226200, China; [email protected] Correspondence: [email protected]; Tel./Fax: +86-21-6443-2140

Academic Editors: Johannes Haybaeck and William Chi-shing Cho Received: 19 July 2016; Accepted: 2 September 2016; Published: 8 September 2016

Abstract: Forkhead box P1 (FOXP1) belongs to a family of winged-helix transcription factors that are involved in the processes of cellular proliferation, differentiation, metabolism, and longevity. FOXP1 can affect cell proliferation and migratory ability in hepatocellular carcinoma (HCC) in vitro. However, little is known about the mechanism of FOXP1 in the proliferation of HCC cells. This study aimed to further explore the function of FOXP1 on the proliferation of HCC cells as well as the relevant mechanism involved. Western blot analysis, tumor xenograft models, and flow cytometry analysis were performed to elucidate the function of FOXP1 in the regulation of cell proliferation in human HCC. We observed that silencing FOXP1 significantly suppressed the growth ability of HCC cells both in vitro and in vivo. In addition, knockdown of FOXP1 induced G1/S phase arrest, and the expression of total and phosphorylated Rb (active type) as well as the levels of E2F1 were markedly decreased at 24 h; however, other proteins, including cyclin-dependent kinase (CDK) 4 and 6 and cyclin D1 did not show noticeable changes. In conclusion, downregulation of FOXP1 inhibits cell proliferation in hepatocellular carcinoma by inducing G1/S phase cell cycle arrest, and the decrease in phosphorylated Rb is the main contributor to this G1/S phase arrest. Keywords: FOXP1; proliferation; hepatocellular carcinoma; cell cycle

1. Introduction Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors, with rising incidence in recent decades [1]. The prognosis of patients diagnosed with HCC remains poor despite progress with surgical and non-surgical therapies. Notably, numerous studies have identified a variety of molecules that are involved in the initiation and progression of HCC [2]. Thus, it is highly desirable to find new biomarkers and molecular targets to develop novel strategies that will be more effective for patients. Forkhead box P1 (FOXP1) is a member of the forkhead box P subfamily, which consists of four members (FOXP1-4) [3]. The FOXP subfamily belongs to the FOX superfamily, which has a highly conserved “fork-head” or “winged-helix” DNA-binding domain (DBD) [4]. FOX proteins are involved in cell cycle progression, proliferation, and differentiation, as well as in metabolism, senescence, survival, and apoptosis [5]. FOXP1 is widely expressed in normal human tissues and a Int. J. Mol. Sci. 2016, 17, 1501; doi:10.3390/ijms17091501

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Int. J. Mol. Sci. 2016, 17, 1501 and senescence, survival,

2 of 10 apoptosis[5]. FOXP1 is widely expressed in normal human tissues and a diverse range of cancers [4,6–10]. A growing body of evidence suggests that FOXP1 plays an important role in tumorigenesis and the progression of suggests several types of solid tumors. FOXP1 diverse range of cancers [4,6–10]. A growing body of evidence that FOXP1 plays an important has been identified as having diverse functions, and can act as either a tumor suppressor or role in tumorigenesis and the progression of several types of solid tumors. FOXP1 has been identified an having oncogene. FOXP1 is overexpressed in diffuse large B-cell lymphoma (DLBCL) and as diverse functions, and can act as either a tumor suppressor or an oncogene. FOXP1 is mucosa-associated lymphoid tissue (MALT) lymphoma through a recurrent chromosomal overexpressed in diffuse large B-cell lymphoma (DLBCL) and mucosa-associated lymphoid tissue translocation T (3;through 14) (p14.1; q32) involving IGHtranslocation and FOXP1,T (3; which is associated with poor (MALT) lymphoma a recurrent chromosomal 14) (p14.1; q32) involving IGH prognosis [11–15]. By contrast, the loss of FOXP1 expression has been observed in human glioma and FOXP1, which is associated with poor prognosis [11–15]. By contrast, the loss of FOXP1 expression [16],been prostate cancer and renal [16], cell carcinoma [18], [17], and the FOXP1 in breast is has observed in [17], human glioma prostate cancer andloss renalofcell carcinoma [18],cancer and the correlated withinlower [19]. High of FOXP1 correlates with an aggressively loss of FOXP1 breastsurvival cancer isrates correlated withexpression lower survival rates [19]. High expression of FOXP1 malignant phenotype and may constitute a novel prognostic factor for HCC [20,21]. Downregulation correlates with an aggressively malignant phenotype and may constitute a novel prognostic factor for of FOXP1 resulted in the suppression of migration reducedofviability of and SMMC-7721 cells as HCC [20,21]. Downregulation of FOXP1 resulted in theand suppression migration reduced viability well as an increase [22]. However, the action FOXP1 inthe theaction proliferation ofin HCC of SMMC-7721 cellsin ascell wellapoptosis as an increase in cell apoptosis [22].ofHowever, of FOXP1 the cells is unknown. proliferation of HCC cells is unknown. In the thepresent presentstudy, study,we weinvestigate investigate effect of FOXP1 on the proliferation of HCC and In thethe effect of FOXP1 on the proliferation of HCC cells cells and seek seek to determine the relevant molecular mechanism. to determine the relevant molecular mechanism.

2. Results 2. Results 2.1. 2.1. FOXP1 Is Frequently Frequently Upregulated Upregulated in in HCC HCC Tissues Tissues We We analyzed analyzed the the expression expression levels levels of of FOXP1 FOXP1 in in primary primary HCC HCC samples. samples. The The results results showed showed that that FOXP1 FOXP1 was was significantly significantly upregulated upregulated in in the the cancer cancer tissues tissues of of62 62matched matchedHCC/normal HCC/normal samples by qRT-PCR samples by Western blot analysis (Figure 1C), qRT-PCR (Figure (Figure 1A,B) 1A,B) and and 24 24 matched matched HCC/normal HCC/normal samples and and aa similar similar result result was was confirmed confirmed in in aa 50-patient 50-patient cohort cohort from The The Cancer Cancer Genome Atlas (TCGA) (Figure (Figure S1A,B). S1A,B).

Figure 1. 1. Forkhead Forkhead box box P1 P1 (FOXP1) (FOXP1) expression expression in hepatocellular hepatocellular carcinoma (HCC) clinical samples. Figure (A) FOXP1 mRNA levels in the 62-patient cohort; The fold fold change change of of FOXP1 FOXP1 levels levels in in paired paired (A) FOXP1 mRNA levels in the 62-patient cohort; (B) The tumor/non-tumorous tissues of the 62-patient cohort; (C) FOXP1 protein levels in 24 pairs of HCC tumor/non-tumorous tissues of the 62-patient cohort; (C) FOXP1 protein of HCC tissues (T) (T) and and their their corresponding corresponding adjacent adjacent noncancerous noncanceroustissues tissues(N). (N). tissues

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2.2.2.2. Silencing HCC Cells Cells in inVitro Vitro SilencingFOXP1 FOXP1Inhibits Inhibitsthe theGrowth Growth Ability Ability of of HCC ToTo proceed qRT-PCR and andWestern Westernblotting blottingwere wereperformed performed proceedwith withthe thesubsequent subsequent research, research, qRT-PCR to to detect the expression of FOXP1 in HCC cell lines. We found that FOXP1 expression was generally high detect the expression of FOXP1 in HCC cell lines. We found that FOXP1 expression was generally in high all ofinthe HCC cell lines 2A). Thus, we selected common Huh7 andand MHCC-97L cells, alltested of the tested HCC cell(Figure lines (Figure 2A). Thus, we selected common Huh7 MHCC-97L both of both which relativerelative high levels of FOXP1, for our study. cells, of express which express high levels of FOXP1, forprimary our primary study.

Figure 2. The effect of FOXP1 on the growth ability of HCC cells in vitro. (A) FOXP1 mRNA and Figure 2. The effect of FOXP1 on the growth ability of HCC cells in vitro. (A) FOXP1 mRNA and protein levels in the HCC cell lines; (B) FOXP1 mRNA and protein levels in Huh7 and MHCC-97L protein levels in the HCC cell lines; (B) FOXP1 mRNA and protein levels in Huh7 and MHCC-97L cells stably transfected with shFOXP1, the scrambled sequence control cells (NC) and untreated cells cells stably transfected with shFOXP1, the scrambled sequence control cells (NC) and untreated cells (MOCK); (C) MTT assays for Huh7 and MHCC-97L cells that were stably transfected with either (MOCK); (C) MTT assays for Huh7 and MHCC-97L cells that were stably transfected with either shFOXP1 or NC. *** p < 0.001; (D) Colony formation assays for Huh7 and MHCC-97L cells that were shFOXP1 or NC. *** p < 0.001; (D) Colony formation assays for Huh7 and MHCC-97L cells that were stably transfected with shFOXP1 or a scrambled sequence control. *** p < 0.001. stably transfected with shFOXP1 or a scrambled sequence control. *** p < 0.001.

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To better understand understand the the function function of of FOXP1 FOXP1 in in HCC, HCC, four four lentiviral lentiviral vectors vectors expressing expressing various various To better short-hairpin short-hairpin RNAs RNAs designed designed to to knockdown knockdown FOXP1 FOXP1 (shFOXP1) (shFOXP1) were were stably stably expressed expressed in in Huh7 Huh7 and and MHCC-97L cells. We selected two efficient hairpins (shFOXP1-1 and shFOXP1-2) that sustained MHCC-97L cells. We selected two efficient hairpins (shFOXP1-1 and shFOXP1-2) that sustained more more 50% reduction of FOXP1 the mRNA and protein (Figure 2B). proliferation Cell proliferation than athan 50%areduction of FOXP1 at theatmRNA and protein level level (Figure 2B). Cell was was detected byMTT the MTT for seven Onsixth the sixth and seventh the number of viable detected by the assayassay for seven days.days. On the and seventh day, day, the number of viable cells cellssignificantly was significantly decreased in Huh7 and MHCC-97L cells stably expressing shFOXP-1 was decreased in Huh7 and MHCC-97L cells stably expressing shFOXP-1 compared with compared with the controls (Figure 2C). Moreover, the colony formation assays confirmed a similar the controls (Figure 2C). Moreover, the colony formation assays confirmed a similar result (Figure 2D). result (Figure 2D). Therefore, these results suggest that FOXP1 downregulation significantly inhibits Therefore, these results suggest that FOXP1 downregulation significantly inhibits HCC cell growth HCC cell growth in vitro. in vitro. 2.3. Knockdown of FOXP1 Decreases Tumorigenicity of HCC HCC Cells Cells in in Vivo Vivo Tumorigenicity of To further clarify the effect of endogenous FOXP1 on tumor growth in vivo, Huh7-lenti-shFOXP1 and Huh7-lenti-control Huh7-lenti-controlcells cellswere were orthotopically inoculated in left thehepatic left hepatic mice. The orthotopically inoculated in the lobe oflobe mice.ofThe tumors tumors in the were after observed after four weeks.the Notably, the tumor was in the liver andliver lungand werelung observed four weeks. Notably, tumor weight was weight remarkably remarkably in the shFOXP1-expressing tumor-bearing mice in comparison to the control decreased indecreased the shFOXP1-expressing tumor-bearing mice in comparison to the control group group (Figure 3A). FOXP1 in the xenograft tumors were analyzed by qRT-PCR and (Figure 3A). FOXP1 proteinprotein levels inlevels the xenograft tumors were analyzed by qRT-PCR and Western Western blotting (Figure 3B). These results indicate that FOXP1 plays an important role in tumor blotting (Figure 3B). These results indicate that FOXP1 plays an important role in tumor formation of formation of HCC and can be a positive regulator of HCC growth. HCC and can be a positive regulator of HCC growth.

The effect effect of of FOXP1 FOXP1 on on the tumorigenicity tumorigenicity of of HCC HCC cells in vivo. (A) Huh7 cells stably Figure 3. The vivo. (A) expressing shFOXP1-1 shFOXP1-1 were injected injected orthotopically orthotopically into into nude nude mice; mice; empty empty vectors vectors were were used used as a control. The The tumors tumors were removed from the nude mice after four weeks. Representative images are shown along with tumors. ** ** p