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Oncogenes and Tumor Suppressors

The Transcription Factor AP4 Promotes Oncogenic Phenotypes and Cisplatin Resistance by Regulating LAPTM4B Expression

Lu Wang, Yue Meng, Jian-Jun Xu and Qing-Yun Zhang
DOI: 10.1158/1541-7786.MCR-17-0519 Published May 2018

Abstract

Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a novel oncogene, whose overexpression is involved in cancer occurrence and progression. However, the mechanism of LAPTM4B transcriptional regulation remains unclear. In this study, the results of transcription factor (TF) profiling plate arrays indicated that AP4 was a potential transcription factor regulating LAPTM4B expression. LAPTM4B was positively correlated with AP4 and they were both associated with poor overall and disease-free survival. Luciferase and electrophoretic mobility shift assay assays confirmed that AP4 directly bound to the polymorphism region of LAPTM4B promoter and modulated its transcription. Functionally, AP4 promoted cell proliferation, migration, invasion, and assisted drug resistance in part through upregulation of LAPTM4B. Taken together, these findings identify LAPTM4B as a direct AP4 target gene and the interaction of AP4 and LAPTM4B plays an important role in breast cancer progression.

Implications: This study demonstrates that AP4 promotes cell growth, migration, invasion, and cisplatin resistance through upregulation of LAPTM4B expression, thus representing an attractive therapeutic target for breast cancer. Mol Cancer Res; 16(5); 857–68. ©2018 AACR.

Introduction

Breast cancer is the most frequently diagnosed malignancy in women worldwide and the second leading cause of cancer-related mortality among females worldwide (1). Despite early detection and enhanced management, breast cancer is expected to account for 15% of total new cancers in China (2). Identification of novel prognostic markers and a thorough understanding of the marker's mechanism will provide a basis for designing therapeutic strategies for breast cancer patients.

LAPTM4B is a newly identified oncogene (NM_018407, Gene ID: 55353) and was first cloned in human hepatocellular carcinoma (HCC) in 2000. LAPTM4B protein has been found to be widely expressed in normal human tissues and upregulated in various types of carcinomas (3). It could promote the proliferation of tumor cells, boost invasion and metastasis, resist apoptosis, initiate autophagy, and assist drug resistance (4). In addition, overexpression of LAPTM4B was significantly correlated with poor prognosis in breast cancer (5), gallbladder cancer (6, 7), ovarian cancer (8), HCC (9–11), gastric cancer (12), and cervical cancer (13) etc. It is noteworthy that the LAPTM4B gene has two alleles named LAPTM4B*1 and LAPTM4B*2 (GenBank numbers AY219176 and AY219177, respectively). Allele *1 contains only one copy of a 19-bp sequence at the 5'UTR in the first exon, whereas the sequence in allele *2 is duplicated and tandemly repeated (14). Previous study showed that LAPTM4B *2/2-type allele was significantly associated with the susceptibility of various adenocarcinoma including breast cancer (15–17), HCC (18, 19), non–small lung cancer (20), gastric cancer (21), cervical cancer (22), colorectal cancer (23), lymphoma (24), gallbladder carcinoma (25), ovarian carcinoma (26), and malignant melanoma (27). Therefore, we hypothesized that if there are some specific transcription factors binding to the LAPTM4B polymorphism region that play important roles in regulating LAPTM4B expression.

In this study, we analyzed the LAPTM4B polymorphism region in the first exon and found the transcription factor AP4 conserved binding sites in the 19-bp polymorphism region. The expression levels of AP4 and LAPTM4B and their prognostic significance were demonstrated in breast cancer. AP4 promoted breast cancer proliferation, migration, invasion, and cisplatin resistance partially by directly upregulating LAPTM4B expression. These effects were associated with cell cycle, EMT, and PI3K/AKT signaling pathways. The results provide strong evidence supporting the expression and functional coupling between AP4 and LAPTM4B and help to explore new therapeutic targets for breast cancer.

Materials and Methods

Computer information analysis and TF profiling plate arrays

The 5′-genomic sequences of LAPTM4B of different species were gained from GenBank. Promoter prediction and analysis for potential transcription factors binding to the polymorphism region of LAPTM4B promoter were performed using UCSC and PROMO (TRANSFAC 8.3) website online programs (http://www.genome.ucsc.edu/ and http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3).

The TF profiling plate arrays (Signosis) were used to search for transcription factors binding to the promoter region of LAPTM4B*1 and LAPTM4B*2 alleles. The promoter probes for the two alleles were located in −200∼+301bp and −200∼+320bp, respectively. The nucleotide sequences are numbered with the transcription start site as +1.

Cell lines and transfection

Human breast cell lines MCF10A, MCF7, and ZR-75-1 were purchased from Cellcook (Guangzhou, China). MDA-MB-231 and T47D cell lines were kindly provided by Dr. Shou Cheng-Chao (Peking University Cancer Hospital & Institute, Beijing, China) and were authenticated by short tandem repeat analysis (Genetic Testing Biotechnology Corporation) in February 2017. The cells were cultured according to the ATCC recommended conditions. They were transfected with siRNA, shRNA, or corresponding empty vectors as described in Supplementary Table S1.

Patients and tissue samples

The tissue samples were obtained from 146 breast cancer patients who were surgically treated in the Beijing Cancer Hospital from January 2008 to December 2010. All the patients underwent primary mastectomies plus axillary lymph node dissection. None of the patients had received adjuvant chemotherapy, immunotherapy, or radiotherapy before. Survival information was available for all patients until March 4, 2017. This study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Peking University School of Oncology. The informed consent was obtained from all participants.

IHC analysis

The expressions of AP4 and LAPTM4B in breast cancer tissues were examined by IHC. The slides were incubated with anti-AP-4 polyclonal antibody (dilution 1:200, Abcam ab28512) and anti-LAPTM4B polyclonal antibody (dilution 1:200, Bioss Inc, bs-6542R) at 4°C overnight. Stained tissue sections were evaluated separately by two pathologists blinded to the clinical parameters. The percentage of positive-staining tumor cells was scored as follows: 0 (<5% positive tumor cells), 1 (5%–50% positive tumor cells), and 2 (>50% positive tumor cells). Staining intensity was scored as follows: 0 (no staining or only weak staining), 1 (moderate staining), and 2 (strong staining). The sum of the staining intensity score and the percentage score was used to define the AP-4 and LAPTM4B expression levels: 0–2, low expression; 3–4, high expression.

Plasmid construction

Stable AP4 (GV341, Ubi-MCS-SV40-puromycin) and LAPTM4B overexpression (GV358, Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin) and control cell lines were created through lentivirus infection. Stable knockdown of AP4 was also established by infection of lentivirus particles (GV248, hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) carrying AP4 shRNA and the target sequence of shAP4 was shown in Supplementary Table S1. They were both constructed by GeneChem. To generate the LAPTM4B gene promoter luciferase reporter plasmid, the truncated LAPTM4B gene upstream sequence was cloned into pGL3-Basic plasmid (Promega). The six truncated plasmids (pGL3-PF1–6) were conserved by our laboratory (28). AP4-binding site inside the LAPTM4B polymorphism region was site-directed mutated by SOE-PCR using modified reverse primer spanning the AP4-binding site (SBS Genetech Co., Ltd). All AP4 specific siRNAs were synthesized by RiboBio and the sequences are shown in Supplementary Table S1.

Dual luciferase assays

For the binding of AP4 to the LAPTM4B promoter, breast cancer cells were planted into 24-well plates in 1 × 105 per well, and incubated for 12 hours in complete medium. Then MCF7, T47D, ZR-75-1, and MDA-MB-231 were cotransfected with 500 ng of LAPTM4B promoter plasmid and 20 ng phRL-CMV plasmid in triplicate. Transfection was carried out using Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer's instructions. At 48 hours after transfection, luciferase activity was measured using the dual-luciferase reporter assay system (Promega). The luciferase value of pGL3-promoter (positive control) was set to 100% and the other constructs were compared with it.

ChIP assays

MCF7 and MDA-MB-231 cells and their derivatives were cultured as described above. For cross-linking, formaldehyde was added to a final concentration of 1%. The reaction was stopped by addition of glycine at a final concentration of 0.125 mol/L. Chromatin was sheared to an average fragment size of 200–1,000 bp by sonication. The sheared chromatin was then immunoprecipitated by using goat polyclonal AP-4 antibody (sc-18593X, Santa Cruz Biotechnology) or normal goat IgG (CR2, Sino Biological Inc). Identification of the captured LAPTM4B promoter fragments was performed by qPCR analysis by using the promoter primers. The sequence of primers was shown in Supplementary Table S1.

EMSA and super-shift assay

The annealed double-strand oligonucleotide derived from the LAPTM4B promoter containing either the wild-type (5′-GGA GCT CCA GCA GCT GGC TGG AGC C-3′) or mutated AP-4 binding site (5′-GGA GCT CCA Gtc aac tGC TGG AGC C-3′) was 3′ end-labeled by biotin. The AP-4-binding site was underlined and the mutated nucleotides were denoted in lowercase. Consensus wild-type and mutant AP-4 probes were synthesized by Viagene and nuclear extracts were prepared by Nuclear and Cytoplasmic Extraction reagents (Viagene). Five micrograms of nuclear protein was mixed for 30 minutes at room temperature with biotin-labeled oligonucleotide using LightShift Chemiluminescent EMSA Kit (Pierce) according to the manufacturer's instructions.

For competition experiments, 100- and 200-fold molar excess of unlabeled competitor probes were preincubated with nuclear extract for 30 minutes on ice before biotin-labeled probes were added. For the super-shift assay, 1 μg and 2 μg of goat anti-human AP-4 polyclonal antibody were added to the binding reaction. Normal goat IgG was the control.

Western blot analysis

Cells were washed twice with ice-cold PBS and lysed using RIPA extraction reagent (TIANGEN). Whole-cell lysates were boiled in lysis buffer containing 5% SDS. Protein concentration was determined by BCA protein assay kit (Pierce). Samples were subjected to SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. The membranes were then incubated with primary antibody at 4°C overnight (antibodies were depicted in Supplementary Table S2) and secondary antibody at room temperature for 1 hour. Signals were detected using enhanced chemiluminescence reagents (Pierce). In all experiments, β-actin was used as an internal control.

Reverse transcription and real-time PCR assay

Total RNA was isolated from cells using TRIzol (Invitrogen) according to manufacturer's instructions. Reverse transcription of 2 μg total RNA was carried out using oligo (dT16) primer. Quantitative real-time PCR was performed on a Roche 480 using SYBR Green Real-time PCR Master Mix (TIANGEN). The sequences of the PCR primers were shown in Supplementary Table S1. All reactions were run in triplicate.

Cell proliferation assay

The cell proliferation assay was performed with Cell Counting Kit-8 (CCK-8; Dojindo). Cells were seeded into 96-well plates at 1 × 103 cells per well and then incubated for 5 days. Each time point, 10 μL CCK-8 solution was added to each well and incubated for 2 hours at 37°C. Then the results were measured with a microplate reader at 450 nm in accordance with the manufacturer's instructions. In colony formation assay, the cells were seeded on 60-mm dishes. After 2 weeks, the colonies were fixed in 4% formaldehyde and stained with 1% crystal violet. Finally, positive colony formation (>50 cells/colony) was counted. Each experiment was repeated in triplicate.

Cell-cycle assay

The cells were collected and fixed in 70% cold ethanol for at least 12 hours at 4 °C. Cells were stained with 50 μg/mL propidium iodide (BD Biosciences) at room temperature for 15 minutes in dark. Cell cycle was performed by FACSCalibur system (BD Biosciences) and analyzed by ModFit 3.0 software (BD Biosciences).

Cell migration and invasion assay

The migration and invasion of breast cancer cells were detected using 24-well transwell cell culture chambers (8.0-μm pore size, Costar). For invasion assay, the insert membranes were precoated with Matrigel (BD Biosciences). The cells were incubated at 37°C for 24 hours (migration assay) and 48 hours (invasion assay). The nonmigratory or noninvasive cells in the top chambers were removed with cotton swabs. The migrated and invaded cells on the bottom membrane surface were fixed in in 4% formaldehydel, air-dried, stained with 1% crystal violet, and then counted under a microscope.

Annexin V apoptosis assay

MCF7 and MDA-MB-231 cells were incubated with different concentration of cisplatin and collected after 48-hour incubation. Apoptosis was analyzed using an PE-Annexin V Apoptosis Detection kit (BD Biosciences) with FACSCalibur flow cytometer (BD Biosciences).

Tumorigenesis in NOD/SCID mice

The MDA-MB-231 cell populations with stable AP4 knockdown, LAPTM4B overexpression, AP4 knockdown+LAPTM4B overexpression, and control vectors were constructed by lentiviral infection and puromycin selection. A total of 20 six-week-old female SCID mice purchased from Beijing HFK Bioscience Co., Ltd were randomly distributed into four groups (5 mice per group). Each mice was injected subcutaneously into the flanks with 100-μL PBS containing 2 × 106 tumor cells. The tumor volume was determined twice every week by measuring the length (l) and width (w) and then calculating the volume as 1/2(w2×l). All animal studies were conducted with the approval of Medical Experimental Animal Care Commission of Peking University School of Oncology.

Statistical analysis

All the statistical analyses were evaluated using the Statistical Software Package for the Social Sciences (SPSS software version 19.0, SPSS), and P values <0.05 were considered to be statistically significant. Data were reported as the means ± SD. Spearman rank correlation was utilized to evaluate the relationship between AP-4 and LAPTM4B expression levels of breast cancer tissues. Survival analysis was performed using the Kaplan–Meier method. A Student t test was used to assess differences between two groups. Differences in cell growth curves and in vivo tumorigenicity were confirmed with repeated-measures ANOVA.

Results

Screen transcription factors binding to LAPTM4B polymorphism region in breast cancer cells

We used the promoter-binding TF profiling plate arrays to explore the transcription factors which could bind to LAPTM4B promoter region in breast cancer cell. Genomic DNA fragments of two alleles of LAPTM4B (LAPTM4B*1: −200bp∼+301bp and LAPTM4B*2: −200bp∼+320bp) were used as TF-bound probes in MCF7 cells (Supplementary Fig. S1). As a result, we screened 10 transcription factors that may bind to the the promoter of LAPTM4B*1 and 24 factors binding to LAPTM4B*2 (Supplementary Fig. S2). Then, we predicted the potential transcription factors binding to 19-bp polymorphism region of LAPTM4B alleles by computer analysis (Supplementary Table S3, S4). As a result, only AP-4 and ELK were selected after matching the result of TF profiling plate arrays. Therefore, AP4 which got the higher relative score was chosen for further study.

AP4 and LAPTM4B are coexpressed in breast cancer and indicate poor survival

We first examined AP-4 and LAPTM4B protein levels in four breast cancer cell lines (MCF7, ZR-75-1, T47D, and MDA-MB-231) and one immortalized breast epithelial cell line MCF10A by Western blot analysis (Fig. 1A). MCF10A exhibited the lowest expression levels of both AP4 and LAPTM4B, while MCF7 and MDA-MB-231 got the higher expression than the other cell lines (Fig. 1B). Thus, LAPTM4B protein expression may be correlated with AP4 expression in these breast epithelial cell lines. Furthermore, we analyzed the mRNA levels of AP4 and LAPTM4B in 1021 breast cancer patients from the TCGA (The Cancer Genome Atlas) database and found that LAPTM4B expression significantly correlates with AP4 at the mRNA level (Fig. 1C). In our study, we examined the expression levels of AP-4 and LAPTM4B in 146 breast cancer tissues by IHC (Table 1; Fig. 1D). As a result, AP-4 was significantly positively correlated with LAPTM4B expression in breast cancer tissues (Table 1). Survival analysis indicated that patients with high levels of both AP4 and LAPTM4B had the poorest outcome in terms of overall survival and disease-free survival (Fig. 1E).

Figure 1.
Figure 1.

AP4 and LAPTM4B are coexpressed in breast cancer. A, Expression of AP4 and LAPTM4B at the protein level in one immortalized breast epithelial cell line and four BC cell lines were determined by Western blotting. B, The protein levels were measured by ImageJ software. The expression levels of AP4 and LAPTM4B in MCF10A were set to 1.0. C, LAPTM4B mRNA expression levels were significantly correlated with AP4 in 1,021 breast cancer patients from the TCGA database. D, 146 breast cancer specimens were analyzed by IHC staining and four representative cases were shown. Original magnification, × 200; scale bar, 200 μm. E, The prognostic value of the combination of AP4 and LAPTM4B was determined in 146 breast cancer cases.

View this table:
Table 1.

The correlation between AP4 and LAPTM4B expressions in breast cancer patients (n = 146)

AP4 induces LAPTM4B transcription in breast cancer cells

To further test whether LAPTM4B is a direct target of AP4, we analyzed the sequence upstream of the LAPTM4B gene. Figure 2A is model chart of the potential AP4-binding sites in the LAPTM4B polymorphism region and luciferase reporter constructs, demonstrating that there is only one AP4-binding site in the polymorphism region of LAPTM4B*1 allele but include two sites of LAPTM4B*2 due to the repetition of 19 bp. The core sequence of the truncated LAPTM4B promoter and the mutated AP4-binding sites were shown in Fig. 2B. Dual-luciferase reporter assay showed the fragment of +10∼+292 (pGL3-PF6) had the highest transcriptional activity among the seven 5′-deleted constructs (Fig. 2C). The transcriptional activities of mutated constructs with AP4-binding site mutation of LAPTM4B*1 allele (pGL3-PF6 mut) were significantly decreased than the wild-type. The transcriptional activities of constructs with only one AP4-binding site mutation of LAPTM4B*2 allele (pGL3-PF7 mut1, pGL3-PF7 mut2) were significantly increased, but became the lowest when the two binding sites of AP4 (pGL3-PF7 mutD) were mutated at the same time (Fig. 2C). The results suggest that AP4 could regulate the transcriptional activity of LAPTM4B gene promoter and may affect the transcription activity of LAPTM4B*2 allele more than LAPTM4B*1.

Figure 2.
Figure 2.

AP4 transcriptionally upregulates LAPTM4B by binding to the LAPTM4B gene promoter. A, The potential AP4-binding sites in LAPTM4B*1 and LAPTM4B*2 alleles and luciferase constructs ranging across the 5′-region were presented. 5′-region is black lines and the transcription start site (TSS) is depicted as the right-point arrow. The gray box indicates the AP4-binding site. Asterisk is referred to the mutated AP4-binding site. B, Sequences containing AP4-binding site and its corresponding mutated form of two LAPTM4B alleles were shown. C, Relative luciferase activities from a series of 5′-deleted constructs and mutated constructs were performed in MCF7, MDA-MB-231, T47D, and ZR-75-1 cells. The CMV promoter-driven Renilla luciferase gene (phRL-CMV) was used as the internal control. D, EMSA assay. Lane 1 was the free probe. Lane 2 was the AP4-containing double-stranded DNA probe and showed a signal shift due to transcription factor binding (shift band). Lanes 3–4 showed that the shift signal can be inhibited from 100-fold, 200-fold excess unlabeled probe. Lanes 5 was AP4 conserved binding site mutated competitor. The shift signal could not be inhibited from mutated probe. E, Super-shift assay. Lane 1 was the free probe. Lanes 2 was bio-probe incubated with nuclear extract. Lanes 3–4 showed that the dsDNA-protein with 1-fold, 2-fold specific anti-AP4 antibodies had slower electrophoretic mobility (super-shift band). Lane 5 was the dsDNA–protein complex with normal goat IgG (control). F, ChIP assay was performed in MCF7 and MDA-MB-231 cells. Precipitated DNA with either anti-AP4 antibody (IP) or normal goat IgG (IgG) and input chromatin (Input) were amplified by PCR with the primers specific for LAPTM4B promoter (a) or a control primer pair (b). G, Relative amounts of AP4 binding to the LAPTM4B promoter were determined by quantitative PCR. H, AP4 shRNAs were introduced into MCF7 and MDA-MB-231 cells for 72 hours. The mRNA and protein levels of AP4 and LAPTM4B were determined by qRT-PCR and Western blotting. I, The expression of AP4 and LAPTM4B at the mRNA and protein levels were examined in MCF7 and MDA-MB-231 cells exposed to AP4 overexpression vector (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

EMSA and super-shift assay showed that transcription factor AP4 could bind to the LAPTM4B polymorphism region specifically in MCF7 cells (Fig. 2D and E). ChIP assay was performed to assess the effect on the direct binding of AP4 to the LAPTM4B promoter and the result showed that anti-AP4 antibody, not goat IgG, specifically (15-fold and 8-fold difference, respectively) immunoprecipitated the LAPTM4B gene promoter in MDA-MB-231 with LAPTM4B*2/2 genotype (29), and MCF7 with LAPTM4B*1/1 genotype (Fig. 2F and G). Taken together, the results suggest that more AP4 protein binds to the LAPTM4B*2 allele than LAPTM4B*1 in its promoter region, which may result that AP4 affected the transcription activity of LAPTM4B*2 allele much more.

To investigate the regulation of LAPTM4B by AP4, an AP4 shRNA vector or AP4 overexpression vector was introduced into MCF7 and MDA-MB-231 cells. AP4 knockdown dramatically reduced the LAPTM4B mRNA and protein levels in both cell lines (Fig. 2H). The results were also confirmed by three siRNA transfection (Supplementary Fig. S3). AP4 overexpression consistently and dramatically upregulated LAPTM4B expression at mRNA and protein levels (Fig. 2I).

AP4 promotes the proliferation of breast cancer cells in vitro and in vivo through LAPTM4B

To explore the roles of LAPTM4B in AP4-mediated breast cancer progression, a rescue experiment was then designed. We knocked down AP4 using shRNA but overexpressed LAPTM4B in MCF7 and MDA-MB-231 cell lines (Fig. 3A). The CCK-8 assay showed that AP4 shRNA noticeably reduced breast cancer cell viability and proliferation; however, LAPTM4B overexpression markedly rescued cell proliferation arrest observed with AP4 depletion (Fig. 3B). The inhibition of colony formation by AP4 shRNA was also antagonized by LAPTM4B overexpression in both cell lines (Fig. 3C). In addition, cell distribution in the cell cycle was determined by flow cytometry. Cells with AP4 knockdown displayed significantly higher frequency at G0–G1 phase in MCF7 and MDA-MB-231 cell lines and a lower frequency at S-phase in MCF7 while overexpression of LAPTM4B partially restored the influence on the G1–S transition in AP4-shRNA cells (Fig. 3D and E). Furthermore, cellular levels of some cell-cycle regulators showed that c-Myc, cyclin E1, and cyclin D1 were significantly decreased in AP4-shRNA cells, but the cyclin-dependent kinase inhibitor p27Kip1(CDKN1B), p21 (CDKN1A), and p16 (CDKN2A) were increased with AP4 knockdown. The effect of these protein levels could also be partly rescued by LAPTM4B overexpression. However, p21 was not affected by LAPTM4B overexpression in MDA-MB-231 cell lines (Fig. 3F).

Figure 3.
Figure 3.

AP4 promotes cell growth through LAPTM4B in vitro and in vivo. A, Cells were transfected with AP4 shRNA with or without LAPTM4B overexpression in MCF7 and MDA-MB-231 cells. Endogenous LAPTM4B expression was indicated by the bottom band (35 kDA) and the top band (38 kDA) represented the exogenous LAPTM4B overexpression. AP4 knockdown decreased the endogenous LAPTM4B expression levels. B, Cell viability of MCF7 and MDA-MB-231 cell lines were determined by CCK-8 assays. C, Cells stably expressing LAPTM4B or AP4 shRNA were cultured for 2 weeks. The number of colonies were counted. D, Flow cytometry analyses were performed, and cell-cycle distributions of MCF7 and MDA-MB-231 cell lines were detected. E, The histograms were analyzed, and the proportions of cells in G0–G1 phase, S-phase, and G2–M phase were shown, respectively. F, The relative expression levels of c-MYC, cyclin E1, cyclin D1, p27, p21, and p16 were examined by Western blotting in MCF7 and MDA-MB-231 cell lines. G, MDA-MB-231 cells with LAPTM4B overexpression, AP4 shRNA, and/or both were injected into mice. The tumor volumes were recorded twice a week. H, The final tumor weights were measured (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Our in vivo data showed that knockdown of AP4 significantly inhibited MDA-MB-231-bearing xenograft growth and overexpression of LAPTM4B accelerated xenograft growth (Fig. 3G). The weight and volume decreased in AP4 knockdown tumors (Fig. 3G and H). Consistent with the in vitro data, AP4-inhibited tumor growth was significantly reversed by LAPTM4B overexpression (Fig. 3G and H). These data collectively suggest that AP4 could promote cell proliferation at least partly by upregulating LAPTM4B.

AP4 promotes breast cancer cell migration and invasion through LAPTM4B by mediating EMT signals and metastasis-related genes

The effect of LAPTM4B on AP4-mediated cell migration and invasion was then studied. The transwell assays showed that AP4 shRNA noticeably reduced cell migration and invasion and LAPTM4B overexpression significantly increased the number of migrated and invaded cells in MCF7 and MDA-MB-231 cell lines (Fig. 4A and B). The inhibition of cell migration and invasion induced by AP4 shRNA was markedly rescued by LAPTM4B overexpression in both cell lines (Fig. 4A and B). Furthermore, AP4 depletion resulted in downregulation of the mesenchymal marker vimentin and N-cadherin expression and upregulation of E-cadherin protein levels in MCF7 and MDA-MB-231 cell lines (Fig. 4C). The expression levels of metastasis-related genes MMP-2 and MMP-9 were also decreased in AP-shRNA cells (Fig. 4C). These effects could be partially restored by LAPTM4B overexpression (Fig. 4C). Therefore, AP4 promotes cell migration and invasion partly by upregulating LAPTM4B through EMT signals and metastasis-related genes.

Figure 4.
Figure 4.

AP4 promotes migration and invasion through LAPTM4B by mediating EMT signals and metastasis-related genes. The effects of AP4 and LAPTM4B on cell migration (A) and invasion (B) were determined by transwell assays in MCF7 and MDA-MB-231 cell lines. C, AP4 modulated the expression of selected EMT markers and metastasis-related proteins via regulating LAPTM4B in breast cancer cell lines. The levels of vimentin, N-cadherin, E-cadherin, MMP-2, and MMP-9 were examined by Western blotting (***, P < 0.001).

AP4 motivates cisplatin resistance partly through LAPTM4B via activating PI3K/AKT pathway

MDR (multidrug resistance, MDR) is a major clinical obstacle in the treatment of cancers. To determine whether AP4 and LAPTM4B can change the chemotherapy sensitivity of breast cancer cells, the inhibition values for cisplatin in MCF7 and MDA-MB-231 cell lines were determined. As shown in Fig. 5A, AP4 shRNA could significantly enhance the chemosensitivity of breast cancer cells to cisplatin, compared with the control groups. Forced overexpression of LAPTM4B reversed the promotion of the chemosensitivity to cisplatin induced by AP4 shRNA (Fig. 5A). LAPTM4B has been shown to motivate multidrug resistance by activation the PI3K/AKT signaling pathway (30). In our study, we detected the effects of PI3K-specific inhibitor on AP4- and LAPTM4B-associated MDR. The results showed that the effects of AP4 and LAPTM4B on MDR were eliminated in the presence of LY294002 (Fig. 5B).

Figure 5.
Figure 5.

AP4 motivates cisplatin resistance partly through LAPTM4B associated with PI3K/AKT pathway. A, The MCF7 and MDA-MB-231 cell lines were exposed to increasing concentration of cisplatin for 48 hours. Cell viability was determined by CCK-8 assays. B, The MCF7 and MDA-MB-231 cell lines were incubated with LY294002 (1 μmol/L) for 48 hours. Cell viability was analyzed by CCK-8 assays. C, The relative expression levels of total AKT, phosphorylations of AKT, and GSK3β in the PI3K/AKT pathway were determined by Western blotting. D, The MCF7 and MDA-MB-231 cell lines were incubated with 20 μmol/L and 40 μmol/L cisplatin, respectively (cisplatin group) and complete medium (control group). After 48-hour incubation, cells were harvested and analyzed by FACSCalibur flow cytometer. E, Column diagrams of apoptotic cells in percentage. F, Cleaved PARP and cleaved caspase-3 were detected by Western blotting in MCF7 and MDA-MB-231 cell lines (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

In addition, the relationship between AP4, LAPTM4B, and the activation of PI3K/AKT signaling pathway was explored. As shown in Fig. 5C, lower levels of phosphorylations of AKT, GSK3β, appeared in AP4 knockdown cells compared with control cells, but no difference of the total AKT protein level was found. The alterations of these proteins involved in PI3K/AKT signaling pathway could also be partly rescued by LAPTM4B restoration. The results also showed that treatment with LY294002 greatly reduced the p-AKT and p-GSK3β in either AP4 knockdown or its controls, and eliminated the elevation of p-AKT and p-GSK3β in LAPTM4B -overexpressing cells (Fig. 5C), implicating the involvement of PI3K/AKT signaling in AP4 and LAPTM4B-motivated MDR. As described above, AP4 and LAPTM4B could influence c-MYC expression in MCF7 and MDA-MB-231 cell lines. We further examined whether the regulation of c-MYC is through PI3K/AKT signaling. As shown in Fig. 5C, c-MYC was significantly decreased in the presence of LY294002 and the regulation by AP4 and LAPTM4B was eliminated.

Chemotherapeutic drugs could induce a series of cellular response that impact on tumor cell apoptosis. As shown in Fig. 5D and E, AP4 knockdown cells were more sensitive to apoptosis in the control group (no cisplatin treatment) or cisplatin group. The effects were rescued significantly by LAPTM4B restoration in MCF7 and MDA-MB-231 cell lines. Moreover, our results herein showed that in the presence of cisplatin, the levels of active forms of caspase-3 and nuclear PARP enhanced in AP4-shRNA cells, but attenuated dramatically with LAPTM4B overexpression (Fig. 5F).

Therefore, it is confirmed that one of the mechanisms for AP4 and LAPTM4B motivation cisplatin resistance is antiapoptosis by activating PI3K/AKT signaling pathway.

Discussion

LAPTM4B, an oncogene, is highly expressed in many solid tumors and inversely associated with overall survival and disease-free survival of patients with breast cancer (31), gastric cancer (32), gallbladder cancer (33), HCC (34), ovarian cancer (35), and cervical cancer (36). Moreover, the polymorphism region in the 5′-UTR of LAPTM4B gene was certified to be associated with tumor susceptibility including breast cancer (15–17). However, the mechanism of the transcriptional regulation of LAPTM4B has been rarely studied. In this study, we identified that transcription factor AP4 could bind to the polymorphism region of LAPTM4B gene and noticeably regulate its expression in breast cancer. Moreover, AP4 could promote breast cancer proliferation, migration, and drug resistance partly through upregulation of LAPTM4B.

Transcription factor AP4 is a member of the basic helix-loop-helix (bHLH) protein family. It was initially shown to activate late viral gene expression from the SV40 enhancer (37). It has been reported that AP4 is upregulated in many types of tumors and linked to poor prognosis in cancer patients (38, 39). We also demonstrated herein that AP4 and LAPTM4B were coexpressed in breast epithelial cell lines and breast tissues, and patients with higher levels of AP4 and LAPTM4B were likely to associated with shorter overall and disease-free survival. In addition, AP4 could bind to the polymorphism region of LAPTM4B promoter and promote LAPTM4B expression in breast cancer cells. Interestingly, luciferase assay showed that the transcriptional activity of LAPTM4B*1 promoter was significantly decreased with AP4-binding site mutation. However, the activity was increased when a single AP4-binding site was mutated in LAPTM4B*2 promoter. It concludes that there may be trans-acting factors playing negative regulatory roles in the polymorphism region of LAPTM4B*2 and further research is needed to clarify it. We further showed that AP4 was capable of inducing cell growth and migration by upregulating LAPTM4B in breast cancer. Conversely, a previous study reported that AP4 could suppress MCF7 cell proliferation by enhancing SHP1 expression and JNK activation (40). It indicates that AP4 may play a dual role in cell proliferation via different pathways. Similar to our results, AP4 has been reported to promote cell growth in HCC (41), colorectal carcinoma (42), and gastric cancer (43). Moreover, it has been proposed that AP4 could activate cell migration and EMT mediated by p53 in MDA-MB-231 cells (44) and mediate a c-MYC–induced EMT in colorectal cancer (45).

Mechanistically, AP4 transcriptionally upregulated LAPTM4B expression to activate c-MYC, p21, and EMT signaling pathway to enhance cell growth and migration in the study. Interestingly, it has been shown that c-MYC could directly regulate AP4 expression and c-MYC-AP4-p21 cascade plays an important role in maintaining cells in a proliferative, progenitor-like state of HCC and colorectal carcinoma (41, 42). Taken together, these results suggest that c-MYC–AP4–LAPTM4B–c-MYC axis may form a feedback loop to participate in cell growth and metastasis.

Recent study revealed that downregulation of AP4 effectively triggered apoptosis and enhanced chemosensitivity of human gastric cancer cells (43). Amplification of LAPTM4B and YWHAZ contributed to anthracycline resistance and recurrence of breast cancer (46). Moreover, LAPTM4B was a marker of resistance to neoadjuvant chemotherapy in HER2-negative breast cancer (47). One of the mechanisms for MDR involves in alternations in the apoptotic response associated with PI3K/AKT signaling. The PPRP motif in the N-terminus of LAPTM4B-35 protein directly binding to PI3K p85 α subunit could strongly phosphorylate AKT and then motivate MDR in HeLa cells (30). In this study, we showed that silencing AP4 induced apoptosis and sensitized cells to cisplatin via activating PI3K/AKT pathway partly by suppressing LAPTM4B. The findings may provide a promising novel strategy for sensitizing chemical therapy of cancers. In addition, AKT could regulate c-MYC through GSK3β in response to mitochondrial stress (48). In our study, we found that AP4 and LAPTM4B may influence c-MYC expression through PI3K/AKT signaling with PI3K inhibitor.

However, we did not find any difference on the regulation of LAPTM4B alleles between MCF7 (LAPTM4B*1/1) and MDA-MB-231 (LAPTM4B*2/2) cell lines. It does not mean that there is no regulation distinction due to different characteristics of cell itself. Thus, the roles of AP4 on the two alleles in the same cell line should be further studied.

In conclusion, this is the first report demonstrating that AP4 is positively correlated with LAPTM4B expression and predicts unfavorable overall and disease-free survival in breast cancer. AP4 tightly controls LAPTM4B gene transcription by binding to the polymorphism region of LAPTM4B promoter. In addition, knockdown of AP4 inhibits proliferation, migration, invasion, and chemotherapy resistance through suppression of LAPTM4B in breast cancer (Fig. 6). Collectively, our data suggest that the newly identified AP4/LAPTM4B axis may play an important role in breast cancer progression and serve as potential therapeutic targets for clinical management.

Figure 6.
Figure 6.

Proposed scheme of molecular basis for effect of AP4/LAPTM4B axis in breast cancer according to our results.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: L. Wang, Y. Meng, Q.Y. Zhang

Development of methodology: L. Wang, Y. Meng, Q.Y. Zhang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Wang, Y. Meng

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Wang, Y. Meng

Writing, review, and/or revision of the manuscript: L. Wang, Q.Y. Zhang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. Wang, Y. Meng, J.J. Xu

Study supervision: J.J. Xu, Q.Y. Zhang

Acknowledgments

This study was supported by National Natural Science Foundation of China (No. 81572910; awarded to Q.Y. Zhang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

  • Received September 19, 2017.
  • Revision received December 7, 2017.
  • Accepted January 2, 2018.
  • Published first January 29, 2018.

References

  1. 1.↵
    1. Siegel RL,
    2. Miller KD,
    3. Jemal A
    . Cancer statistics, 2015. CA Cancer J Clin 2015;65:5–29.
    OpenUrlCrossRefPubMed
  2. 2.↵
    1. Chen W,
    2. Zheng R,
    3. Baade PD,
    4. Zhang S,
    5. Zeng H,
    6. Bray F,
    7. et al.
    Cancer statistics in China, 2015. CA Cancer J Clin 2016;66:115–32.
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Kasper G,
    2. Vogel A,
    3. Klaman I,
    4. Grone J,
    5. Petersen I,
    6. Weber B,
    7. et al.
    The human LAPTM4b transcript is up-regulated in various types of solid tumours and seems to play a dual functional role during tumour progression. Cancer Lett 2005;224:93–103.
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Meng Y,
    2. Wang L,
    3. Chen D,
    4. Chang Y,
    5. Zhang M,
    6. XU J-J,
    7. et al.
    LAPTM4B: an oncogene in various solid tumors and its functions. Oncogene 2016;35:6359–65.
    OpenUrl
  5. 5.↵
    1. Xiao M,
    2. Jia S,
    3. Wang H,
    4. Wang J,
    5. Huang Y,
    6. Li Z
    . Overexpression of LAPTM4B: an independent prognostic marker in breast cancer. J Cancer Res Clin Oncol 2013;139:661–7.
    OpenUrl
  6. 6.↵
    1. Zhou L,
    2. He XD,
    3. Yu JC,
    4. Zhou RL,
    5. Shan Y,
    6. Rui JA
    . Overexpression of LAPTM4B-35 attenuates epirubucin-induced apoptosis of gallbladder carcinoma GBC-SD cells. Surgery 2011;150:25–31.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. Zhou L,
    2. He XD,
    3. Chen J,
    4. Cui QC,
    5. Qu Q,
    6. Rui JA,
    7. et al.
    Overexpression of LAPTM4B-35 closely correlated with clinicopathological features and post-resectional survival of gallbladder carcinoma. Eur J Cancer 2007;43:809–15.
    OpenUrlCrossRefPubMed
  8. 8.↵
    1. Yin MZ,
    2. Li C,
    3. Li X,
    4. Lou G,
    5. Miao B,
    6. Liu X,
    7. et al.
    Over-expression of LAPTM4B is associated with poor prognosis and chemotherapy resistance in stages III and IV epithelial ovarian cancer. J Surg Oncol 2011;104:29–36.
    OpenUrlCrossRefPubMed
  9. 9.↵
    1. Yang H,
    2. Lin M,
    3. Xiong FX,
    4. Yang Y,
    5. Nie X,
    6. McNutt MA,
    7. et al.
    Combined lysosomal protein transmembrane 4 beta-35 and argininosuccinate synthetase expression predicts clinical outcome in hepatocellular carcinoma patients. Surg Today 2011;41:810–7.
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. Yang H,
    2. Xiong FX,
    3. Qi R,
    4. Liu Z,
    5. Lin M,
    6. Rui J,
    7. et al.
    LAPTM4B-35 is a novel prognostic factor of hepatocellular carcinoma. J Surg Oncol 2010;101:363–9.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Peng C,
    2. Zhou RL,
    3. Shao GZ,
    4. Rui JA,
    5. Wang SB,
    6. Lin M,
    7. et al.
    Expression of lysosome-associated protein transmembrane 4B-35 in cancer and its correlation with the differentiation status of hepatocellular carcinoma. World J Gastroenterol 2005;11:2704–8.
    OpenUrlPubMed
  12. 12.↵
    1. Zhang H,
    2. Tian B,
    3. Yu H,
    4. Yao H,
    5. Gao Z
    . LAPTM4B-35 protein as a potential therapeutic target in gastric cancer. Tumour Biol 2014;35:12737–42.
    OpenUrl
  13. 13.↵
    1. Meng F,
    2. Luo C,
    3. Hu Y,
    4. Yin M,
    5. Lin M,
    6. Lou G,
    7. Zhou R
    . Overexpression of LAPTM4B-35 in cervical carcinoma: a clinicopathologic study. Int J Gynecol Pathol 2010;29:587–93.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Liu X,
    2. Zhou R,
    3. Zhang Q,
    4. Zhang Y,
    5. Shao G,
    6. Jin Y,
    7. et al.
    Identification and characterization of LAPTM4B encoded by a human hepatocellular carcinoma-associated novel gene. Beijing Da Xue Xue Bao 2003;35:340–7.
    OpenUrlPubMed
  15. 15.↵
    1. Fan M,
    2. Liu Y,
    3. Zhou R,
    4. Zhang Q
    . Association of LAPTM4B gene polymorphism with breast cancer susceptibility. Cancer Epidemiol 2012;36:364–8.
    OpenUrlPubMed
  16. 16.↵
    1. Li X,
    2. Kong X,
    3. Chen X,
    4. Zhang N,
    5. Jiang L,
    6. Ma T,
    7. et al.
    LAPTM4B allele *2 is associated with breast cancer susceptibility and prognosis. PLoS One 2012;7:e44916.
    OpenUrlPubMed
  17. 17.↵
    1. Shaker O,
    2. Taha F,
    3. Salah M,
    4. El-Marzouky M
    . LAPTM4B gene expression and polymorphism as diagnostic markers of breast cancer In Egyptian patients. J Med Biochem 2015;34:393.
    OpenUrl
  18. 18.↵
    1. Zhai G,
    2. Yang H,
    3. Ji X,
    4. Xiong F,
    5. Su J,
    6. McNutt MA,
    7. et al.
    Correlation of LAPTM4B polymorphisms with hepatocellular carcinoma in Chinese patients. Med Oncol 2012;29:2744–9.
    OpenUrlPubMed
  19. 19.↵
    1. Wang S,
    2. Zhang QY,
    3. Zhou RL
    . Relationship between LAPTM4B gene polymorphism and susceptibility of primary liver cancer. Ann Oncol 2012;23:1864–9.
    OpenUrlCrossRefPubMed
  20. 20.↵
    1. Tang H,
    2. Tian H,
    3. Yue W,
    4. Li L,
    5. Li S,
    6. Gao C,
    7. et al.
    LAPTM4B polymorphism is associated with nonsmall cell lung cancer susceptibility and prognosis. Oncol Rep 2014;31:2454–60.
    OpenUrl
  21. 21.↵
    1. Liu Y,
    2. Zhang QY,
    3. Qian N,
    4. Zhou RL
    . Relationship between LAPTM4B gene polymorphism and susceptibility of gastric cancer. Ann Oncol 2007;18:311–6.
    OpenUrlCrossRefPubMed
  22. 22.↵
    1. Meng F,
    2. Song H,
    3. Luo C,
    4. Yin M,
    5. Xu Y,
    6. Liu H,
    7. et al.
    Correlation of LAPTM4B polymorphisms with cervical carcinoma. Cancer 2011;117:2652–8.
    OpenUrl
  23. 23.↵
    1. Cheng XJ,
    2. Xu W,
    3. Zhang QY,
    4. Zhou RL
    . Relationship between LAPTM4B gene polymorphism and susceptibility of colorectal and esophageal cancers. Ann Oncol 2008;19:527–32.
    OpenUrlCrossRefPubMed
  24. 24.↵
    1. Sun L,
    2. Zhang QY,
    3. Liu Y,
    4. Qian N
    . Relationship between human novel gene LAPTM4B polymorphism and susceptibility of lymphoma. Zhong Liu Fang Zhi Yan Jiu 2007;4:245–8.
    OpenUrl
  25. 25.↵
    1. Yang H,
    2. Zhai G,
    3. Ji X,
    4. Xiong F,
    5. Su J,
    6. McNutt MA
    . Correlation of LAPTM4B polymorphisms with gallbladder carcinoma susceptibility in Chinese patients. Med Oncol 2012;29:2809–13.
    OpenUrl
  26. 26.↵
    1. Xu Y,
    2. Liu Y,
    3. Zhou R,
    4. Meng F,
    5. Gao Y,
    6. Yang S,
    7. et al.
    LAPTM4B polymorphisms is associated with ovarian cancer susceptibility and its prognosis. Jpn J Clin Oncol 2012;42:413–9.
    OpenUrlCrossRefPubMed
  27. 27.↵
    1. Zhang M,
    2. Zhou R,
    3. Xu J,
    4. Zhang Q
    . Relationship between LAPTM4B gene polymorphism and susceptibility of malignant melanoma in Chinese patients. Transl Oncol 2014;7:638–43.
    OpenUrl
  28. 28.↵
    1. Zhang M,
    2. Xu JJ,
    3. Zhou RL,
    4. Zhang QY
    . cAMP responsive element binding protein-1 is a transcription factor of lysosomal-associated protein transmembrane-4 Beta in human breast cancer cells. PLoS One 2013;8:e57520.
    OpenUrlPubMed
  29. 29.↵
    1. Li X,
    2. Kong X,
    3. Chen X,
    4. Zhang N,
    5. Jiang L,
    6. Ma T,
    7. et al.
    LAPTM4B Allele *2 is associated with breast cancer susceptibility and prognosis. PLoS One 2012;7:e44916.
    OpenUrlPubMed
  30. 30.↵
    1. Li L,
    2. Wei XH,
    3. Pan YP,
    4. Li HC,
    5. Yang H,
    6. He QH,
    7. et al.
    LAPTM4B: a novel cancer-associated gene motivates multidrug resistance through efflux and activating PI3K/AKT signaling. Oncogene 2010;29:5785–95.
    OpenUrlCrossRefPubMed
  31. 31.↵
    1. Li S,
    2. Wang L,
    3. Meng Y,
    4. Chang Y,
    5. Xu J,
    6. Zhang Q,
    7. et al.
    Increased levels of LAPTM4B, VEGF and survivin are correlated with tumor progression and poor prognosis in breast cancer patients. Oncotarget 2017;8:41282–93.
    OpenUrl
  32. 32.↵
    1. Zhang H,
    2. Tian B,
    3. Yu H,
    4. Yao H,
    5. Gao Z
    . LAPTM4B-35 protein as a potential therapeutic target in gastric cancer. Tumour Biol 2014;35:12737–42.
    OpenUrl
  33. 33.↵
    1. Zhou L,
    2. He XD,
    3. Yu JC,
    4. Zhou RL,
    5. Yang H,
    6. Qu Q,
    7. et al.
    Overexpression of LAPTM4B promotes growth of gallbladder carcinoma cells in vitro. Am J Surg 2010;199:515–21.
    OpenUrlCrossRefPubMed
  34. 34.↵
    1. Yang H,
    2. Xiong FX,
    3. Lin M,
    4. Yang Y,
    5. Nie X,
    6. Zhou RL
    . LAPTM4B-35 overexpression is a risk factor for tumor recurrence and poor prognosis in hepatocellular carcinoma. J Cancer Res Clin Oncol 2010;136;275–81.
    OpenUrlCrossRefPubMed
  35. 35.↵
    1. Yang Y,
    2. Yang H,
    3. McNutt MA,
    4. Xiong F,
    5. Nie X,
    6. Li L,
    7. et al.
    LAPTM4B overexpression is an independent prognostic marker in ovarian carcinoma. Oncol Rep 2008;20:1077–83.
    OpenUrlPubMed
  36. 36.↵
    1. Meng F,
    2. Tan S,
    3. Liu T,
    4. Song H,
    5. Lou G
    . Predictive significance of combined LAPTM4B and VEGF expression in patients with cervical cancer. Tumour Biol 2015;37:4849–55.
    OpenUrl
  37. 37.↵
    1. Mermod N,
    2. Williams TJ,
    3. Tjian R
    . Enhancer binding factors AP4 and AP-1 act in concert to activate SV40 late transcription in vitro. Nature 1988;332:557–61.
    OpenUrlCrossRefPubMed
  38. 38.↵
    1. Buechler S
    . Low expression of a few genes indicates good prognosis in estrogen receptor positive breast cancer. BMC Cancer 2009;9:243.
    OpenUrlCrossRefPubMed
  39. 39.↵
    1. Hu BS,
    2. Zhao G,
    3. Yu HF,
    4. Chen K,
    5. Dong JH,
    6. Tan JW
    . High expression of AP4 predicts poor prognosis for hepatocellular carcinoma after curative hepatectomy. Tumor Biol 2013;34:271–6.
    OpenUrl
  40. 40.↵
    1. Amin S,
    2. Kumar A,
    3. Nilchi L,
    4. Wright K,
    5. Kozlowski M
    . Breast cancer cells proliferation is regulated by tyrosine phosphatase SHP1 through c-jun N-terminal kinase and cooperative induction of RFX-1 and AP-4 transcription factors. Mol Cancer Res 2011;9:1112–25.
    OpenUrlAbstract/FREE Full Text
  41. 41.↵
    1. Ge Z,
    2. Zhang B,
    3. Bu X,
    4. Wang Y,
    5. Xiang L,
    6. Tan J
    . Molecular mechanism of activating protein-4 regulated growth of hepatocellular carcinoma. Tumour Biol 2014;35:12441–7.
    OpenUrl
  42. 42.↵
    1. Jung P,
    2. Menssen A,
    3. Mayr D,
    4. Hermeking H
    . AP4 encodes a c-MYC-inducible repressor of p21. Proc Natl Acad Sci U S A 2008;105:15046–51.
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    1. Liu X,
    2. Zhang B,
    3. Guo Y,
    4. Liang Q,
    5. Wu C,
    6. Wu L,
    7. et al.
    Down-regulation of AP-4 inhibits proliferation, induces cell cycle arrest and promotes apoptosis in human gastric cancer cells. PLoS One 2012;7:e37096.
    OpenUrlPubMed
  44. 44.↵
    1. Chen S,
    2. Chiu SK
    . AP4 activates cell migration and EMT mediated by p53 in MDA-MB-231 breast carcinoma cells. Mol Cell Biochem 2015;407:57–68.
    OpenUrl
  45. 45.↵
    1. Jackstadt R,
    2. Roh S,
    3. Neumann J,
    4. Jung P,
    5. Hoffmann R,
    6. Horst D,
    7. et al.
    AP4 is a mediator of epithelial–mesenchymal transition and metastasis in colorectal cancer. J Exp Med 2013;210:1331–50.
    OpenUrlAbstract/FREE Full Text
  46. 46.↵
    1. Li Y,
    2. Zou L,
    3. Li Q,
    4. Haibe-Kains B,
    5. Tian R,
    6. Li Y,
    7. et al.
    Amplification of LAPTM4B and YWHAZ contributes to chemotherapy resistance and recurrence of breast cancer. Nat Med 2010;16:214–8.
    OpenUrlCrossRefPubMed
  47. 47.↵
    1. de Ronde JJ,
    2. Lips EH,
    3. Mulder L,
    4. Vincent AD,
    5. Wesseling J,
    6. Nieuwland M,
    7. et al.
    SERPINA6, BEX1, AGTR1, SLC26A3, and LAPTM4B are markers of resistance to neoadjuvant chemotherapy in HER2-negative breast cancer. Breast Cancer Res Treat 2013;137:213–23.
    OpenUrlCrossRefPubMed
  48. 48.↵
    1. Gleyzer N,
    2. Scarpulla RC
    . Concerted action of PGC-1-related coactivator (PRC) and c-MYC in the stress response to mitochondrial dysfunction. J Biol Chem 2016;291:25529–41.
    OpenUrlAbstract/FREE Full Text
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The Transcription Factor AP4 Promotes Oncogenic Phenotypes and Cisplatin Resistance by Regulating LAPTM4B Expression
Lu Wang, Yue Meng, Jian-Jun Xu and Qing-Yun Zhang
Mol Cancer Res May 1 2018 (16) (5) 857-868; DOI: 10.1158/1541-7786.MCR-17-0519
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