ZNF133 is a potent suppressor in breast carcinogenesis through dampening L1CAM, a driver for tumor cell invasion

doi:10.21203/rs.3.rs-2249338/v1

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Research Article

ZNF133 is a potent suppressor in breast carcinogenesis through dampening L1CAM, a driver for tumor cell invasion

https://doi.org/10.21203/rs.3.rs-2249338/v1

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Background

Because of the complexity and heterogeneity, therapeutic effect of breast cancer varies in each subtype, which is classified based on the molecular markers for estrogen or progesterone receptors and human epidermal growth factor 2. Thus, novel comprehensive and precise molecular indicators in breast carcinogenesis are urgently in need.

Methods

The expression of ZNF133 in breast cancer tissues was detected by immunohistochemical staining. The interaction between KAP1 complex and ZNF133 was identified by affinity purification-coupled mass spectrometry. The regulatory mechanisms were validated by luciferase reporter assay and co-immunoprepitation. The target genes of ZNF133 was determinged by chromatin immunoprecipitation-based deep sequencing. Gain-or-loss-of-function assays were used to identify the function and underlying mechanisms of ZNF133 in breast cancer. Cancer cell proliferation, invasion, and tumorigenesis of breast cancer cells were analyzed using cell counting assays, colony formation, transwell, and xenograft tumor models.

Results

Here we report that ZNF133, a zinc-finger protein, is negatively associated with advanced pathological staging and poor survival of breast carcinomas. Moreover, ZNF133 is a transcription repressor, physically associated with the KAP1 complex and transcriptionally represses a cohort of genes including L1CAM that are critically involved in cell proliferation and motility. We demonstrate that the ZNF133/KAP1 complex inhibits the proliferation and invasion of breast cancer cells in vitro and suppresses breast cancer growth and metastasis in vivo through dampening the transcription of L1CAM.

Conclusion

Our study ascertains the value of ZNF133 and L1CAM level in the diagnosis and prognosis of breast cancer, contributes to the deeper understanding of the regulation mechanism of ZNF133 for the first time, and provides a new therapeutic strategy and precise intervention target for breast cancer.

ZNF133

KAP1

L1CAM

Breast cancer

Progression

Metastasis

Worldwide, breast cancer is the most commonly diagnosed malignancy and the commonest cause of cancer related mortality in women. Poor prognosis of breast cancer patients are generally associated with metastatic disease(1). Risk profiles and treatment strategies for breast cancer depend mainly on three major subtypes categorized according to estrogen or progesterone receptor expression and ERBB2 gene amplification(2, 3). However, lots of molecular players and mechanisms behind the complexity of its clinical behavior remain unknown(4). Novel molecules are expected to be unraveled in breast cancer. Thus, in-depth molecular characterizations play critical roles in the current anticancer research era and the future of cancer diagnosis and prognosis evidently rely on tremendously precise molecular index(5).

Zinc finger proteins (ZFPs) constitute the largest individual family of transcription factors in tetrapod vertebrates(6). In humans, approximately one third of about 800 ZFPs contain the Krüppel associated box (KRAB). Most members of the KRAB-ZFPs family have been identified as crucial regulators of diverse cellular processes including embryonic development, cell differentiation, cell proliferation, apoptosis, neoplastic transformation and cell cycle regulation(7). Dysfunction of Zinc-finger protein 133 (ZNF133), a member of the KRAB-ZFPs family, is also implicated in various pathological states including malignancies. It had been reported that ZNF133 was involved in posterior polymorphous corneal dystrophy (PPCD), inflammatory bowel diseases, and alcoholism disease(8–10). In addition, aberrant expression of ZNF133 was involved in benign bone tumors, osteosarcoma and neuroblastoma(11, 12). For example, ZNF133 was proposed as a potential protein involved in repression of S100A4, which was associated with increased metastatic capacity of various types of cancer cells(13). In 2020, ZNF133 was reported to be overexpressed after irradiation in patients who survived a childhood malignancy, thus was considered as a candidate of radiation-sensitive genes for childhood tumor(14). However, the molecular mechanism underlying the role of ZNF133 in tumorigenesis is still poorly understood.

L1 cell adhesion molecular (L1CAM) is a transmembrane glycoprotein. Its full-length form is composed of a long ectodomain that comprises six immunoglobulin-like (Ig) domains followed by five fibronectin type III repeats, a single transmembrane domain and a relatively short highly conserved cytoplasmic domain(15). Initially L1CAM was found in neurons in central system, known for its role in neural development, by its ability to regulate neurite outgrowth, fasciculation, cell adhesion, cell migration, myelination, and cell survival(16, 17). Subsequent studies lately unveiled that L1CAM was overexpressed in primary tumors and metastases of various types of cancers such as glioma, melanoma, ovarian, colon, breast and pancreatic cancers(18–21), and was associated with poor prognosis. In these cancers, tumor cells tend to use the same mechanism involving L1CAM as neurons do to possess an increased migratory and proliferative potential. In pancreatic cancer cells, for instance, downregulation of L1CAM inhibits proliferation, invasion and arrests cell cycle progression(22). In 2018, studies from Memorial Sloan Kettering Cancer Center (MSK) revealed that L1CAM was necessary for metastatic colonization in multiple organs such as lung, bone, brain and liver in breast cancer(23). Hence, L1CAM is a valuable diagnostic marker and an attractive target for the therapy of breast cancer.

The control of L1CAM expression in cancers is complex and is affected by both transcriptional and epigenetic mechanisms. Positive regulation of L1CAM was reported for the transcription factors β-catenin and SLUG in colon, endometrial and pancreatic cancer cells(19, 24), and negative regulation of L1CAM was found for the AR and REST in triple-negative breast cancers and small cell lung cancer(25, 26). Nevertheless, the molecular mechanism underlying the regulation of L1CAM expression in breast carcinogenesis remains poorly explored.

In this study, we report that L1CAM expression is suppressed by ZNF133, a novel KRAB zinc-finger protein in breast cancer cells. We demonstrate that ZNF133 recruits the KAP1 complex to the L1CAM promoter and represses L1CAM transcription. We demonstrate that the ZNF133 inhibits the proliferation and invasive potential of breast cancer cells in vitro and suppresses breast cancer growth and metastasis in vivo. These data support a role for ZNF133 as a novel transcription repressor and a potential tumor suppressor in breast cancer.

Tissue microarray (TMA) and immunohistochemistry

The clinical significance of ZNF133 expression in breast cancer patients was analyzed using TMAs obtained from Shanghai Outdo Biotech Co., Ltd (China) that contained 40 breast tumor tissues and adjacent normal tissues. Shanghai Outdo Biotech Co., Ltd also provided patients’ information, including gender, age, pathological grade, and TNM stage. Use of the TMAs complied with relevant regulations, and was approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. Antigen retrieval was performed by incubating the samples in sodium citrate solution (0.01 mol/L, pH 6.0) buffer at high pressure for 15 min. Subsequently, endogenous peroxidase activity was blocked using 3% hydrogen peroxide, the TMAs were incubated with anti-ZNF133 antibody (1:200) at 4°C overnight, and then incubated with secondary antibody for 1 h at room temperature. For immunohistochemistry quantification, all samples were blind scored by three independent observers. The average score was calculated for each tumor sample.

Gene Construction And Lentivirus Production

The cDNAs for ZNF133 were purchased from WZ Biosciences. The cDNAs for L1CAM were purchased from Sino Biological. The cDNA for ZNF133 was amplified by PCR and ligated into XbaI/SwaI sites of a pCDHO-CMV-FLAG vector, NOTI/EcoRV sites of a pCDNA3.1 with one tag of FLAG, or KpnI/SmaI sites of a Gal4-DBD-3XFLAG vector. All clones were confirmed by DNA sequencing. The Gal4-DBD-3XFLAG, Gal4-SV40-LUC, Gal4-TK-LUC, Gal4-AdML-LUC and renilla plasmids were from Dr. Yongjie Ma (Tianjin Medical University Cancer Institute and Hospital, China). The pGEX-4T-1-KAP1 were from Dr. Fanbiao Meng (Tianjin Medical University Cancer Institute and Hospital, China). The pGEX-4T-1-HDAC1/HDAC2/RBBP4/RBBP7 were from Dr. Lin Shan (Capital Medical University, China). The 384 bp fragment from L1CAM promoter and its motif mutant were generated from Azenta Life Sciences. The pLKO.1 lentiviral RNA interference (RNAi) expression system was used to construct lentiviral short hairpin RNA (shRNA) for genes. The sequences of shRNA used in this study included the following: shSCR: 5’-CCTAAGGTTAAGTCGCCCTCG-3’, shZNF133-1: 5’-GCAACCTTCGGATTCCTTATA-3’, shZNF133-2: 5’-CACCTCACCTTACATCAAATG-3’, shKAP1: 5’-GAGAATTATTTCATGCGTGAT-3’, shL1CAM: 5’-CCACTTGTTTAAGGAGAGGAT-3’. For viral packaging, the lentiviral plasmid pCDHO-CMV-ZNF133, pCDHO-CMV-L1CAM, pLKO.1-shZNF133, pLKO.1-shKAP1 or pLKO.1-shL1CAM, together with psPAX2 and pMD2.G were co-transfected into the packaging cell line HEK293T using PEI co-precipitation at 10:5:5 mg (for a 10-cm dish). The transfection medium containing PEI and plasmid mixture was replaced with fresh complete medium after incubation for 6 h. Viral supernatants were collected 48 h later, clarified by filtration, and concentrated by ultracentrifugation. The concentrated virus was used to infect 5 × 10⁵ cells (20–30% confluent) in a 60 mm dish with 5 µg/ml polybrene. Infected cells were selected with puromycin and/or neomycin. For the re-expressing or re-silencing L1CAM experiments, the level of L1CAM expression was controlled by creating stable clones of cells that were expressing different levels of L1CAM, and the clones with L1CAM levels close to original L1CAM level were chosen for phenotype experiments.

Cell Culture And Transfection

MCF-7, MDA-MB-231, and HEK293T cells were obtained from the American Type Culture Collection (ATCC) and maintained according to ATCC recommendations. Transient transfections of expression plasmids in MCF-7, and 293T cells were carried out using poly (ethylene imine) (PEI) (Sigma) according to the manufacturer’s recommendations. Transfections of expression plasmids in MDA-MB-231 cells were carried out using Lipofectamine 3000 (Invitrogen) as the recommendations.

Reagents And Antibodies

Anti-FLAG M2 affinity gel (A2220), and FLAG peptide (F3290) were purchased from Sigma. Protein A/G PLUS-agarose (sc-2003) was purchased from Santa Cruz. Puromycin was purchased from Invitrogen, and neomycin was from Gibco. Crystal violet was purchased from Solarbio. D-luciferin was from Genomeditech. Antibodies used were as follows: anti-ZNF133 (Abcam, ab237736, 1:200 for IHC, 1:100 for IF and 1:1,000 for WB); anti-FLAG (MBL, M185-3L, IP and 1:8,000 for western blot); anti-fibronectin (Proteintech, 15613-1-AP, 1:1,000 for WB); anti-E-cadherin (Proteintech, 20874-1-AP, 1:1,000 for WB); anti-N-cadherin (Proteintech, 22018-1-AP, 1:1,000 for WB); anti-GAPDH (Proteintech, 60004-1-Ig, 1:8,000 for WB); anti-KAP1 (Proteintech, 66630-1-Ig, ChIP and 1:1,000 for WB)/(Proteintech, 15202-1-AP, IP and 1:1,000 for WB); anti-HDAC1 (Proteintech, 10197-1-AP, IP and 1:1,000 for WB); anti-HDAC2 (Proteintech, 12922-3-AP, IP and 1:1,000 for WB); anti-RBBP4 (Proteintech, 20364-1-AP, IP and 1:1,000 for WB); anti-RBBP7 (Proteintech, 20365-1-AP, 1:1,000 for WB); anti-HP1 (Proteintech, 11831-1-AP, IP and 1:1,000 for WB); anti-L1CAM (Abcam, ab270455, 1:1,000 for WB); anti-ERK (Proteintech, 11257-1-AP, 1:1,000 for WB); anti-p-ERK (Proteintech, 28733-1-AP, 1:1,000 for WB); anti-β-actin (Sigma, A5316, 1:5,000 for western blot).

Fluorescence Confocal Microscopy

MCF-7, T47D, or MDA-MB-231 cells growing on six-well chamber slides were washed with PBS, fixed in 4% (w/v) paraformaldehyde for 20 min, permeabilized with 0.1% (v/v) Triton X-100 in PBS for 10 min, blocked with 3% BSA, and incubated with appropriate primary antibodies followed by staining with Alexa Fluor 488-coupled secondary antibodies (Life Technologies, 1:150). The cells were were washed four times, and a final concentration of 0.1 mg/mL DAPI (Sigma) was included in the final washing to stain nuclei. Images were visualized with a fluorescence microscope (Zeiss Imager Z2).

Luciferase Reporter Assay

HEK293T and MCF-7 cells in 24-well plates were transfected with luciferase reporter, renilla, and indicated expression constructs. The amount of DNA in each transfection was kept constant by addition of empty vector. Thirty-six hours after transfection, the firefly and renilla luciferase were assayed according to the manufacturer’s protocol (Promega), and the firefly luciferase activity was normalized to that of renilla luciferase. Each experiment was performed in triplicate and repeated at last three times.

Immunopurification And Mass Spectrometry

HEK293T cells stably expressing FLAG-ZNF133 were washed twice with cold PBS, scraped, and collected by centrifugation at 1500 × g for 5 min. Cellular extracts were prepared by incubating the cells in lysis buffer containing protease inhibitor cocktail (Roche). Anti-FLAG immunoaffinity columns were prepared using anti-FLAG M2 affinity gel (Sigma) following the manufacturer’s suggestions. Cell lysates were obtained from about 5 × 10⁸ cells and applied to an equilibrated FLAG column of 1-ml bed volume to allow for adsorption of the protein complex to the column resin. After binding, the column was washed with cold PBS plus 0.1% Nonidet P-40. FLAG peptide (Sigma) was applied to the column to elute the FLAG protein complex as described by the vendor. Fractions of the bed volume were collected and resolved on NuPAGE 4–12% Bis-Tris gel (Invitrogen), silver-stained using Pierce silver stain kit, and subjected to LC-MS/MS (Q-Exactive HF X, BGI) sequencing.

Co-immunoprecipitation And Western Blotting

Cellular lysates were prepared by incubating the cells in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.3% NP-40, 2 mM EDTA) containing protease inhibitor cocktail (Roche) for 40 min at 4°C, followed by centrifugation at 12,000 × g for 15 min at 4°C. The protein concentration of the lysates was determined by the BCA protein assay kit (Pierce) according to the manufacturer’s protocol. For immunoprecipitation, 500 µg of protein was incubated with 2 µg specific antibodies for 12 h at 4ºC with constant rotation; 50 µl of 50% protein A or G agarose beads was added and incubated for an additional 3 h at 4ºC. Beads were then washed five times using the lysis buffer. Between washes, the beads were collected by centrifugation at 1000 × g for 3 min at 4ºC. The precipitated proteins were eluted from the beads by resuspending the beads in 2 × SDS-PAGE loading buffer and boiling for 10 min. The resultant materials from immunoprecipitation or cell lysates were resolved using SDS-PAGE gels and transferred onto nitrocellulose membranes. For routine western blotting, cells were washed twice with cold PBS, then lysed in buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycer-ophosphate, 1 mM sodium vanadate, 1 mg/ml leupeptin, 1 mM phenylmethyl-sulfonylfluoride). Equal amounts of protein were loaded onto SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) blotting membranes. Membranes were incubated with appropriate antibodies for 1 h at room temperature or overnight at 4°C followed by incubation with a secondary antibody. Immunoreactive bands were visualized using western blotting Luminol reagent (Santa Cruz) according to the manufacturer’s recommendation.

Gst Pull-down Assay

GST fusion constructs were expressed in BL21 E. coli bacteria, and crude bacterial lysates were prepared by sonication in TEDGN (50mM Tris-HCl, pH 7.4, 1.5mM EDTA, 1mM dithiothreitol, 10% (v/v) glycerol, 0.4M NaCl) in the presence of the protease inhibitor mixture. In vitro transcription and translation experiments were done with rabbit reticulocyte lysate (TNT systems, Promega) according to the manufacturer’s recommendation. Briefly, equal amounts of GST fusion proteins were immobilized on 50 µl of 50% glutathione-Sepharose 4B slurry beads (Amersham Biosciences) in 0.5 ml of GST pull-down binding buffer (10 mM HEPES, pH 7.6, 3 mM MgCl₂, 100 mM KCl, 5 mM EDTA, 5% glycerol, 0.5% CA630). After incubation for 1 h at 4°C with rotation, beads were washed three times with GST pull-down binding buffer and resuspended in 0.5 ml of GST pull-down binding buffer before adding 10 µl of in vitro transcribed/translated proteins for 2 h at 4°C with rotation. The beads were then washed three

times with binding buffer. The bound proteins were eluted by boiling in 30 µl of 2 × sample loading buffer and resolved on SDS-PAGE.

Fast Protein Liquid Chromatography

Cellular lysates of MCF-7 stably expressing FLAG-ZNF133 were prepared by incubating the cells in lysis buffer containing protease inhibitor cocktail (Roche). Lysates were then applied to an 850 × 20 mm Superose 6 size exclusion column (Amersham Biosciences) that was equilibrated with PBS and calibrated with protein standards (blue dextran, 2000 kDa; thyroglobulin, 669 kDa; ferritin, 440 kDa; aldolase, 158 kDa; all from Amersham Biosciences). The column was eluted at a flow rate of 0.5 ml/min and fractions were separately collected.

Chip Sequencing

MCF-7 cells stably expressing FLAG-ZNF133 were maintained in DMEM supplemented with 10% FBS. Approximately 5 × 10⁷ cells were used for each ChIP-seq assay. The ChIP procedure was performed as described in the qChIP below. The chromatin DNA was precipitated by monoclonal antibodies against FLAG. The DNA was purified with the Qiagen PCR purification kit. In-depth whole-genome DNA sequencing was performed by the BGI Corporation, China.

Qchip

MCF-7 cells were crosslinked using 1% formaldehyde for 10 min at room temperature and quenched by the addition of glycine to a final concentration of 125 mM for 5 min. The fixed cells were resuspended in lysis buffer (1% SDS, 5 mM EDTA and 50 mM Tris-HCl, pH 8.1) in the presence of protease inhibitors, then subjected to 30 cycles (30-s on and off) of sonication (SCIENTZ-II D) to generate chromatin fragments of 300–500 bp in length. Lysates were diluted in buffer containing 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8.1) and protease inhibitors. For IP, the diluted chromatin was incubated with specific antibodies for 12 h at 4°C with constant rotation, 50 µL of 50% (vol/vol) protein A/G Sepharose beads were then added, and the incubation was continued for an additional 2 h. Beads were washed with the following buffers: TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl and 20 mM Tris-HCl, pH 8.0); TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl and 20 mM Tris-HCl, pH 8.0); TSE III (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl, pH 8.0) and TE (1 mM EDTA and 10 mM Tris-HCl, pH 8.0). The pulled-down chromatin complex and input were de-crosslinked at 55°C for 12 h in elution buffer (1% SDS and 0.1 M NaHCO₃). The eluted DNA was purified with the QIAquick PCR Purification Kit. qChIPs were performed using Power SYBR Green PCR Master Mix and ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA). The sequences of the primers used are listed as following: L1CAM, 5′-TCTACCCTGCCCTTCTCTCC-3′ (forward) and 5′-CTCACCTTCTCCCTGTTCCG-3′ (reverse); NPAS4, 5′-CTGTCGCATGTCTAGGGCAG-3′ (forward) and 5′-CACCGTCCAGGCTCTCTAAC-3′ (reverse); VGF, 5′-CTAGCTCGCTCCGGCTTCAG-3′ (forward) and 5′-CAATCGTCGGGCGTCCTT-3′ (reverse); SYNDIG1L, 5′-TTGACCCCAGTTCATCAGCC-3′ (forward) and 5′-TTGCACTTCGTCCCTCCCTA-3′ (reverse); CADM3, 5′-TTGAGAGCTGGCTGCCTTAC-3′ (forward) and 5′-TGGAGAGCTCAAAGCACAGG-3′ (reverse); SYP, 5′-GCGCGTACCTGATTCACCAC-3′ (forward) and 5′-CCTGGGCTGTTCCAACGA-3′ (reverse); CNTNAP2, 5′-AATCCCCAACTTCAGCACCG-3′ (forward) and 5′-AAAGGGAGACTGAGCCGAC-3′ (reverse); PRKD1, 5′-TGCTGAATGACGCCATCTGT-3′ (forward) and 5′-GTCCACACCCAGTACTGCTC-3′ (reverse); PCNA, 5′-GAGCAAAGAGCCCTGGAACA-3′ (forward) and 5′-TTCGCGCCAAAGTCACAAAG − 3′ (reverse).

Quantitative Rt-pcr Analysis

Total cellular RNAs were isolated from samples with the Trizol reagent (Invitrogen). First strand cDNA synthesis with the Reverse Transcription System (TransGen Biotech). Quantitation of all gene transcripts was done by qPCR using Power SYBR Green PCR Master Mix and an ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA) with the expression of GAPDH as the internal control. The primer pairs used were: L1CAM, 5′-TGCTCATCCTCTGCTTCATC-3′ (forward) and 5′-TCCTCGTTGTCACTCTCCA-3′ (reverse); NPAS4, 5′-CCCAGCACTGCCACGTTCCC-3′ (forward) and 5′- CCCGGAGTTGAGCTAGGGCCA-3′ (reverse); VGF, 5′-AAGGATGACGGCGTACCAGA-3′ (forward) and 5′-TGCCTGCAACAGTACCGAG-3′ (reverse); SYNDIG1L, 5′- GGCGGGACAGGCTCTGA-3′ (forward) and 5′-AGCAGCGGGTTCTGTAGTTC-3′ (reverse); CADM3, 5′-CGGAGAGGGTGAGAAATGG-3′ (forward) and 5′-CGAGAGTAAATCTGTTGGGTTG-3′ (reverse); SYP, 5′-TCTGGTGATGTTTTACGCTAC-3′ (forward) and 5′-AATCCTCAGGCTTAACTTGC-3′ (reverse); CNTNAP2, 5′- CAAGTGTTCAGGTTGTGTCAAGAA-3′ (forward) and 5′-AGCAGGGTATAATGTGGTGAGGA-3′ (reverse); PRKD1, 5′-CGCACATCATCTGCTGAACT-3′ (forward) and 5′-CTTTCGGTGCACAACGTTTA-3′ (reverse); PCNA, 5′-AAAAGCCACTCCACTGTCT-3′ (forward) and 5′-CTCTACAACAAGGGGCACAT-3′ (reverse) ; GAPDH, 5′-TCCTCCTGTTTCATCCAAGC-3′ (forward) and 5′-TAGTAGCCGGGCCCTACTTT-3′ (reverse).

Proliferation Assay

Cells were plated in triplicate in 12-well plates at 1 × 10⁵ cells per well in 1.5 mL of medium. After days, as indicated in experiments, wells were washed twice with PBS to remove dead cells, and then the entire contents of the well were trypsinized. Cell number was determined using a hemocytometer. Each experiment was performed in triplicate and repeated at last threetimes.

Colony Formation Assay

MCF-7 or MDA-MB-231 cells were maintained in culture media in 6 or 12-well plate for 14–20 days, fixed with 4% paraformaldehyde, stained with 0.1% crystal violet for colony observation, and counted using light microscope. Each experiment was performed in triplicate and repeated at last three times.

Cell Invasion Assay

The transwell invasion assay was performed using the transwell chamber (BD biosciences). Stably infected MDA-MB-231 cells were cultured in medium with 10% FBS at 37°C without CO². Cells were deprived in serum-free medium for 18 h, and harvested. These cells were washed three times in PBS and resuspended in serum-free culture medium. Afterwards, 1 × 10⁵ of these cells in 0.3 ml of serum-free media were plated onto the matrigel-coated upper chamber of the transwell. The upper chamber was then transferred into a well containing 0.5 ml of media supplemented with 10% FBS and incubated for 5 h. Cells may actively migrate from the upper to the lower side of the filter due to FBS as attractant. Cells on the upside were removed using cotton swabs, and the invasive cells on the lower side were fixed, stained with 0.1% crystal violet solution, and counted using light microscope. Each experiment was performed in triplicate and repeated at last three times.

In vivo metastasis

The MDA-MB-231-Luc-D3H2LN cell line (MDA-MB-231 cell line engineered to stably express firefly luciferase) (Xenogen Corporation) was infected with lentiviruses carrying ZNF133 or/and L1CAM, or lentiviruses carrying ZNF133 shRNA, or/and L1CAM shRNA. These cells were inoculated into the left abdominal mammary fat pad (5 × 10⁶ cells) of 6-week-old severe immunodeficient female NCG mice (T001475, GemPharmatech). Mice, according to body equal completely randomized design, were divided into groups each with six before injected. Sample size estimate was based on xenograft assays from literatures. Mice in which tumors did not form would be removed from the study. Tumors were observed in all the mice injected with cells. For bioluminescence imaging, mice were anesthetized and given 200 µg/g of D-luciferin in PBS by intraperitoneal injection. Fifteen minutes after injection, bioluminescence images were obtained with a charge-coupled device camera (IVIS SPECTRUM; Caliper life science). Bioluminescence from relative optical intensity was defined manually, and data was expressed as photon flux (photons/s/cm2/Steradian) and normalized to background photon flux which was defined from a relative optical intensity drawn over a mouse that was not given an injection of D-luciferin. The measurement and data processing were done by an investigator blinded to the treatment procedure. All animals were killed at the end of the experiment. Animal handling and procedures were approved by the Animal Ethical and Welfare Committee (AEWC) of Tianjin Medical University Cancer Institute and Hospital (NSFC-AE-2022199).

Statistical analysis

Data from biological triplicate experiments are presented with error bar as mean ± S.D. Two-tailed unpaired Student’s t-test was used for comparing two groups of data unless otherwise noted. Statistical significance was considered at a value of P < 0.05.

A lower expression level of ZNF133 predicts a poorer prognosis in patients with breast cancer

In an effort to unravel the novel and intricate molecular mechanisms underlying the development and progression of breast cancer and to further explore the molecular function of KRAB-ZFPs, we performed Kaplan–Meier survival analysis (http://kmplot.com/analysis/) of public data sets. We found that higher ZNF133 expression was associated with a better relapse-free survival of breast cancer patients (Fig. 1A). Inquiry of public dataset for ZNF133 expression using the Gene expression-based Outcome for Breast cancer Online (GOBO) tool (http://co.bmc.lu.se/gobo/gsa.pl) supported the notion that the level of ZNF133 expression was negatively correlated with the histological staging of the tumors (Fig. 1B). Moreover, the expression of ZNF133 was lower in each breast cancer subtype than in normal tissue (Fig. 1C). Consistently, the body map from Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/) showed that the median level of ZNF133 expression in breast cancer is lower than in normal samples (Fig. 1D). The dot plot showed the gene expression profile across all tumor samples and paired normal tissues (Fig. 1E). Likewise, we analyzed the ZNF133 protein level in clinical specimens from the online dataset of the Human Protein Atlas (https://www.proteinatlas.org/). In the database, the higher expression of ZNF133 indicated longer overall survival time (Fig. 1F). To further confirm the clinical relevance of ZNF133 expression, we analyzed the mRNA expression of ZNF133 in the Oncomine database (https://www.oncomine.org/). Using the TCGA breast cancer datasets, we found lower ZNF133 expression in the subtypes of invasive breast carcinoma tissues than noncancerous tissues (Fig. 1G). The level of ZNF133 expression was higher in normal patients than other four individual breast cancer stages analyzed from the TCGA database in UALCAN analysis portal (http://ualcan.path.uab.edu/index.html) (Fig. 1H).

Next, we performed immunohistochemical staining of human tissue arrays containing 40 breast carcinoma samples paired with normal mammary tissues. We found that ZNF133 was mainly detected in the nucleus and significantly downregulated in breast carcinomas compared with adjacent noncancerous tissues (paired-samples t test, p < 0.001) (Fig. 2A). Remarkably, low ZNF133 expression significantly correlated with advanced pathological grade (p < 0.001) (Fig. 2B). Likewise, we also observed that ZNF133 displayed a positively strong expression in normal breast tissues, and a weak expression in breast cancer (Fig. 2C), based on clinical specimens from the Human Protein Atlas. The above data indicated that ZNF133 is associated with good prognosis and appears to have negative effect in breast cancer progression.

Znf133 Inhibits Proliferation And Invasion Of Breast Cancer Cells

To confirm this speculation, we firstly tested the effect of the ZNF133 on the proliferation of breast cancer cells in vitro. To this end, MCF-7 and MDA-MB-231 cells were infected with lentiviruses carrying ZNF133 expression or ZNF133 shRNA, and the number of cells was examined. Our results showed that ZNF133 overexpression had a significant inhibitory effect on both MCF-7 and MDA-MB-231 cells, whereas knockdown of ZNF133 promoted the proliferation of these cells (Figure S1A). In addition, colony formation assays in MCF-7 and MDA-MB-231 cells showed that overexpression of ZNF133 was associated with a decreased colony number and knockdown of ZNF133 resulted in an increase in colony number (Figure S1B). These experiments support a role for ZNF133 in the inhibition of cell proliferation.

Metastases occur frequently in patients with breast cancer and are associated with significant morbidity and exceptionally poor survival. To investigate the role of ZNF133 in the invasion and metastasis of breast cancer, we first performed transwell cell invasion and tested the effect of ZNF133 on the invasive potential of breast cancer cells. MDA-MB-231 cells were infected with lentiviruses carrying ZNF133 or ZNF133 shRNA. Our results showed that overexpression of ZNF133 was associated with a decrease in the invasive potential and knockdown of ZNF133 led to an increase in the invasive potential of cells (Figure S1C). In addition, epithelial–mesenchymal transition (EMT) is an early event in cell invasion, we then measured the expression of epithelial/mesenchymal markers by western blotting in MCF-7 and MDA-MB-231 cells. The results showed that overexpression of ZNF133 resulted in induction of epithelial protein markers (E-cadherin) and reduction of mesenchymal markers (N-cadherin and Fibronectin) (Figure S1D). Conversely, depletion of ZNF133 resulted in the reduction of epithelial markers and induction of mesenchymal markers (Figure S1D). These results support a role for ZNF133 in the regulation of the invasive potential of breast cancer cells.

Znf133 Is A Transcription Repressor

ZNF133 is mapped to chromosome 20p11.23 and consists of ten exons. The predicted molecular weight of ZNF133 is 73.33 kDa. Bioinformatics analysis indicates that ZNF133 harbors an N-terminal KRAB domain and eleven C2H2 zinc fingers (Figure S2A). Amino-acid sequence alignment reveals that the human ZNF133 is highly similar with its homologs in Pan troglodytes, Canis lupus familiaris, and Rattus norvegicus (Figure S2B). Phylogenetic analysis also indicates that ZNF133 is an evolutionarily well-conserved gene (Figure S2C), indicating a crucial physiological role of ZNF133.

Immunofluorescent imaging of ZNF133 in MCF-7, T47D and MDA-MB-231 cells indicate that ZNF133 is primarily localized in the nucleus (Fig. 3A). To confirm the expression of full-length ZNF133 protein, FLAG-tagged ZNF133 (FLAG-ZNF133) expression plasmids were transfected into MCF-7 and MDA-MB-231 cells and analyzed by western blotting with a monoclonal antibody against FLAG. The results showed that ZNF133 is a protein with a molecular weight of ~ 75 kDa (Fig. 3B).

As stated above, the KRAB-ZFP family proteins have been implicated in transcriptional regulation. To explore the transcriptional activity of ZNF133, we fused the full length of ZNF133 to the C-terminus of Gal4 DNA-binding domain and analyzed the transcriptional activity of the fused construct in HEK293T and MCF-7 cells. We utilized three different Gal4-driven luciferase reporter systems, which differed in basal promoter elements (Fig. 3C). The results showed that ZNF133 significantly inhibits the reporter activity in a dose-dependent manner in all of the three reporter systems in both HEK293T and MCF-7 cells (Fig. 3D), suggesting that ZNF133 is involved in transcription repression. Meanwhile, overexpression of FLAG-ZNF133 did not affect the activity of Gal4-driven reporter in MCF-7 cells (Fig. 3E), suggesting that ZNF133 must physically bind to DNA to exert its transcriptional activity.

Znf133 Is Physically Associated With The Kap1 Complex

To further elucidate the molecular mechanism underlying ZNF133-mediated transcription repression, we then interrogated the ZNF133 interactome in vivo by affinity purification-coupled mass spectrometry. FLAG-ZNF133 was stably expressed in HEK293T cells and the whole cell extracts were prepared and subjected to affinity purification using an anti-FLAG affinity gel. Mass spectrometric analysis indicates that ZNF133 was co-purified with KAP1, HDAC1/2, RBBP4/7 and HP1, all components of the KAP1 complex (Fig. 4A).

To confirm the interaction between ZNF133 and the KAP1 complex in breast cancer cells, protein extracts from MCF-7 and MDA-MB-231 cells overexpressing FLAG-ZNF133 were immunoprecipitated with the anti-FLAG followed by immunoblotting with antibodies against KAP1, HDAC1/2, RBBP4/7 and HP1. The results showed that components of KAP1 complex could be efficiently co-immunoprecipitated by ZNF133 (Fig. 4B). Reciprocally, IP with antibodies against components of the KAP1 complex followed by IB with antibodies against FLAG also confirmed the interaction of ZNF133 with the KAP1 complex (Fig. 4C). Consistently, analysis of FLAG-ZNF133 affinity eluate by fast protein liquid chromatography (FPLC) with Superose 6 columns and a high salt extraction and size exclusion approach in MCF-7 cells stably expressing FLAG-ZNF133 was performed. We found a multiprotein complex containing ZNF133, KAP1, HDAC1/2, RBBP4/7 and HP1 (Fig. 4D). Moreover, glutathione S-transferase (GST) pull-down assays with purified GST-fused KAP1, HDAC1/2, RBBP4/7 and FALG-ZNF133 revealed that ZNF133 was capable of interacting directly with KAP1, but not with other components of the KAP1 complex (Fig. 4E). Taken together, these results support the specific interaction between ZNF133 and KAP1 complex.

Identification Of Genome-wide Transcriptional Targets For The Znf133

To explore the biological significance of the physical interaction of ZNF133, we performed chromatin immunoprecipitation-based deep sequencing (ChIP-seq) to analyze the genome-wide transcriptional targets of ZNF133. In these experiments, ChIP experiments were performed in MCF-7 cells stably expressing FLAG-ZNF133. Then ZNF133 associated DNAs were amplified using nonbiased conditions, labeled, and then sequenced using HiSeq2500. Using MACS (version2) with a p value cutoff of 10^− 3, we identified 2,907 ZNF133-specific binding peaks, and totally 1122 genes in the promoter cluster targeted by ZNF133 were identified. The distribution of specific binding peaks was 42.98% promoter, 29.73% intron, 3.79% exon, 21.71% distal intergenic, 0.14% 5′ UTR5, 1.10% 3′ UTR3, and 0.55% downstream (≤ 3 kb) (Fig. 5A). Additionally, the peaks’ chromosome distribution is shown in Fig. 5B. The 1122 genes targeted by ZNF133 were then classified into various cellular biological processes using the Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/) database. These biological processes include the development of dendrite, perikaryon and neuronal cell body, as well as cell proliferation and cycle pathways (Fig. 5C). Furthermore, we analyzed the genomic binding signatures for ZNF133 and identified the most significant binding motif CTGTCCATGGTCCTGA (Fig. 5D).

By using specific antibodies against FLAG, KAP1, or isotypic normal IgG, Quantitative ChIP (qChIP) analysis in MCF-7 and MDA-MB-231 cells stably expressing FLAG-ZNF133 showed a strong enrichment of ZNF133 and KAP1 on the promoters of selected genes including L1CAM, NPAS4, VGF, SYNDIG1L, CADM3, SYP, CNTNAP2, PRKD1, PCNA that represent each of the classified pathways of these genes, (Fig. 5E), validating the ChIP-seq results. Meanwhile, measurement of the mRNA expression of the selected genes by real-time RT-PCR in MCF-7 and MDA-MB-231cells indicates that the expression of these genes decreased when ZNF133 was overexpressed (Fig. 5F). The above results indicated that ZNF133 recruited KAP1 on target gene promoters in breast cancer cells to repress the gene transcription.

Transcription Repression Of L1cam By Znf133/kap1 Complex

Among the identified genes, L1CAM had been reported correlated with aggressive tumor progression of patients with breast cancer and was present at increased levels in primary tumors and metastases of various types of cancer(18, 27–29), indicating the importance of transcriptional regulation of L1CAM in breast carcinogenesis and a potential of being a therapeutic target against metastatic breast cancer. We found that ZNF133, HDAC1 (GSM3499149), and HDAC2 (ENCFF000QOA) had similar peak locations on the promoter of L1CAM (Fig. 6A), supporting the notion that ZNF133, HDAC1, and HDAC2 physically interact and are functionally linked. What’s more, we searched the proximal promoter of the human L1CAM gene and identified one site in the promoter region that matched the consensus ZNF133-binding element (Fig. 6A), further proving that L1CAM is a potential target of ZNF133.

We next cloned the 384 bp peak fragment containing ZNF133-binding element from L1CAM promoter to construct a luciferase reporter to test whether ZNF133 was capable of repressing the L1CAM promoter driven luciferase reporter (L1CAM-Luc). MCF-7 cells were then co-transfected with L1CAM-Luc and ZNF133 expression plasmid. The results showed that ZNF133 repressed the activity of L1CAM promoter only when CTGTCCATGGTCCTGA motif existed, but not the mutant L1CAM luciferase reporter (Fig. 6B). Additionally, ZNF133 was no longer able to repress L1CAM-Luc activity when KAP1 was knocked down (Fig. 6C), further supporting the targeting of L1CAM gene by the ZNF133/KAP1 complex.

To further confirm the transcription repression of L1CAM by the ZNF133, ZNF133 was overexpressed or knocked down in MCF-7 cells, and western blotting analysis was performed. The results showed that upregulation of ZNF133 was associated with a reduced expression of L1CAM, whereas downregulation of ZNF133 was accompanied by an elevated expression of L1CAM (Fig. 6D). As the subsequent extracellular signal-related kinase 1/2 (ERK1/2) activation was implicated in L1CAM-mediated regulation, we analyzed the phosphorylation of ERK and found that ZNF133 inhibited the intrinsic activation of ERK1/2 simultaneously (Fig. 6D), indicating that ZNF133 may restrain cancer cell progression via the L1CAM/ERK1/2 signaling pathway. Consistently, real-time RT-PCR assay showed that the reduced level of L1CAM mRNA was responded to ZNF133 overexpression, which could be reversed when KAP1 was depleted in MCF-7 cells (Fig. 6E).

Moreover, interrogation of the public dataset for ZNF133, KAP1 and L1CAM expression, we found a negative correlation in mRNA level of ZNF133 and L1CAM, as well as a positive correlation of ZNF133 and KAP1 (Fig. 6F). To further extend our observations to a clinicopathologically relevant setting, stratification of patient groups based on the inverse expression of ZNF133 and L1CAM or the co-expression of ZNF133 and KAP1 was performed in TCGA database, indicating a better survival of breast cancer patients in ZNF133 high and L1CAM low level, or ZNF133 and KAP1 high level (though not significant) (Fig. 6G). Collectively, these results clearly showed that the ZNF133/KAP1 complex potentially represses the transcription of L1CAM in breast cancer.

Role Of L1cam In Promoting Breast Cancer Progression

In order to consolidate the significant role of L1CAM in breast cancer development and progression(18, 23), we first analyzed the overall survival time of patients suffering breast cancer with online clinical Human Protein Atlas dataset, confirming that the higher expression of L1CAM indicated shorter overall survival time (Figure S3A). In addition, in the GOBO dataset, the level of L1CAM expression was positively associated with tumor histological staging (Figure S3B) and the highest in the basal subtype of breast cancer with the strongest metastasis potential (Figure S3C).

In an effort to verify the potential effect of L1CAM on the proliferation and invasion of breast cancer cells, MCF-7 or MDA-MB-231 cells were infected with lentivirus carrying L1CAM expression vector or shRNA. Cell proliferation and invasion were analyzed using cell counting, colony formation and transwell assays, respectively. The results showed that L1CAM overexpression promoted cell proliferation and increased the number of invasive cells, while knockdown of L1CAM significantly inhibited cell proliferation and invasion (Figure S3D-F). What’s more, the results of western blotting showed that the E‑cadherin protein (epithelial marker) was inhibited and N‑cadherin and Fibronectin protein (mesenchymal marker) were induced by L1CAM in MCF-7 and MDA-MB-231 cells, inversely knockdown of L1CAM inhibited the EMT progress (Figure S3G). With all the above results, we confirmed that L1CAM promoted proliferation, invasion and EMT in breast cancer cells.

ZNF133/KAP1 complex suppresses the proliferation and invasion of breast cancer cells through inhibition of L1CAM in vitro

To investigate the role of the ZNF133/KAP1-L1CAM axis in the development and progression of breast cancer, MCF-7 and MDA-MB-231 cells were infected with lentiviruses carrying ZNF133 and/or L1CAM or infected with lentiviruses carrying ZNF133 shRNA and/or L1CAM shRNA, and cell proliferation was examined by cell counting assays and colony formation assays. Our results showed that the proliferation inhibitory effect of ZNF133 overexpression was rescued by simultaneous overexpression of L1CAM to the original level, and the promoted breast cancer cell proliferation by depletion of ZNF133 could be abrogated by co-knockdown of L1CAM (Fig. 7A-B, S4A-B). Furthermore, lentiviruses carrying ZNF133 or concurrently with KAP1 shRNA were infected in MCF-7 and MDA-MB cells and colony formation experiment was performed. The results showed that the effect of ZNF133 overexpression on colony number could be offset by knockdown of KAP1 in both cell lines (Fig. 7C-D, S4C-D), supporting that ZNF133 inhibited cell proliferation via association with KAP1 complex. Taken together, these results presented a role for ZNF133/KAP1 complex in the inhibition of cell proliferation through downregulation of L1CAM expression.

On the other hand, to investigate whether or not the ZNF133/KAP1-L1CAM axis played a crucial role in regulating the invasive potential of breast cancer cells in vitro, we measured the expression of epithelial/mesenchymal markers in MCF-7 and MDA-MB-231 cells. Based on alteration of the EMT marker protein levels, the results showed that the inhibitory effect on EMT by ZNF133 was rescued by recovery of L1CAM to the original level, while the positive effects for EMT progress by knockdown of ZNF133 was attenuated by co-knockdown of L1CAM (Fig. 7E, S4E). What’s more, transwell cell invasion assay proved that the reduced invasion ability of MDA-MB-231 cells associated with ZNF133 overexpression was offset by overexpression of L1CAM, whereas the increase in the invasive potential by ZNF133 knockdown was partially offset by co-knockdown of L1CAM (Fig. 7F). Meantime, the effect on the decreased invasive potential by ZNF133 overexpression was attenuated by simultaneous knockdown of KAP1 (Fig. 7G). Collectively, these results indicated that ZNF133 inhibited the metastasis of breast cancer in vitro through its interaction with the KAP1 complex and repression of target genes including L1CAM.

ZNF133 restrains the growth and metastasis of breast cancer in vivo

To investigate the in vivo role of ZNF133 against the growth and metastasis of breast cancer, MDA-MB-231 cells stably expressing firefly luciferase (MDA-MB-231-Luc-D3H2LN) were infected with lentiviruses carrying ZNF133 or/and L1CAM, or carrying ZNF133 shRNA or/and L1CAM shRNA. These cells were then orthotopically implanted onto the abdominal mammary fat pad (n = 6) of 6-week-old female severe immunodeficiency NCG (NOD/ShiLtJGpt-Prkdc^em26Cd52Il2rg^em26Cd22/Gpt) mice. The growth/dissemination of tumors was monitored weekly by bioluminescence imaging with IVIS imaging system. Tumor metastasis was measured by quantitative bioluminescence imaging after 8 weeks. Metastatic events were defined as any detectable luciferase signal above background and away from the primary tumor site. The results showed that ZNF133 overexpression inhibited while knockdown of ZNF133 promoted the growth of the primary tumor and metastasis of the MDA-MB-231-Luc-D3H2LN tumors to lung, liver, intestine, or spleen (Fig. 8A-C). Remarkably, simultaneous overexpression of L1CAM counteracted the ZNF133 overexpression-associated suppression of the growth of primary tumors and metastases, while growth and metastases were significantly inhibited in mice carrying shZNF133 + shL1CAM tumors compared with shZNF133 (Fig. 8A-C). Above all, these results supported that ZNF133 could suppress the growth of breast cancer by inhibiting L1CAM and thus may be a promising approach for breast cancer treatment (Fig. 8D).

In this study, we reveal that zinc-finger protein, ZNF133, which is a repressive transcription factor, interacting with KAP1 complex to inhibit the expression of L1CAM, an oncogene in breast cancer, suppresses the breast cancer development and progression in vitro and in vivo.

ZNF133 is an evolutionarily conserved gene and can be detected in all tissues. Several studies suggested diverse function of ZNF133 in various malignancies. ZNF133 has been identified as a novel amplified gene in the neuroblastoma cell line(11) and an up-regulated protein in osteosarcoma tissues(12). However, studies on several cell lines, including the breast cancer cells, revealed that ZNF133 was implicated in repression of S100A4 protein which is associated with the bad prognostic outcome of cancer(13). In the current study, we show that the expression of ZNF133 is progressively lost during breast cancer progression, and represses the proliferation and invasive potential of breast cancer cells in vitro and in vivo. Although it is currently unknown about the genetic background of the aberrant down-regulation of ZNF133 in breast cancer development, it is clear that the ZNF133 is a biomarker for the diagnosis and prognosis of breast cancer, and a potential therapeutic pointcut for breast cancer.

L1CAM was reported to be associated with a shorter disease-free, overall survival and impaired prognosis in the breast cancer, based on serious analysis on L1CAM expression in primary breast cancers using the combination of western blotting, DNA-microarray analysis and immunohistochemistry(18, 23, 27, 30, 31). Therefore, understanding of the regulation of L1CAM expression is of great importance for breast cancer prevention and intervention. However, limited information about the negative regulation of its expression in breast cancer is available. It has just been reported that L1CAM could be repressed by androgen receptor (AR) in triple-negative breast cancer (TNBC). Overexpression of AR leads to the loss of L1CAM expression and concomitant binding of AR to binding sites in the L1CAM promoter region in TNBC cells (26). Here, we report that L1CAM can be transrepressed by ZNF133 in both MCF-7 and MDA-MB-231 cells. We confirm that ZNF133 does so through recognizing a CTGTCCATGGTCCTGA motif in the promoter of L1CAM with the KAP1 repressive complex, adding to our knowledge of molecular mechanisms on L1CAM expression regulation. However, whether other transcriptional regulators on L1CAM promoter exist in breast cancer cells remains to be investigated, and how different regulators might be coordinated in the regulation of L1CAM expression in a specific cell lineage or subtypes is still unknown. Therefore, it is valuable to further recognize the abnormalities, genetic or/and epigenetic, of transcriptional regulators associated with L1CAM expression to control breast carcinogenesis.

Physiologically, the tissue expression for ZNF133 is ubiquitous, and especially enhanced in nervous system, such as cerebellar hemisphere, neurons, and oligodendrocytes (Bgee, https://bgee.org/; and tissue specific cell type information in Human Protein Atlas, https://www.proteinatlas.org/humanproteome/tissue+cell+type). L1CAM is also involved in the mammalian nervous stem where they regulate diverse aspects of brain development. L1CAM plays a crucial role in regulation of synaptic development and plasticity, as well as axo-axonic innervation(16, 17). The expression distributions of ZNF133 and L1CAM have suggested the distinctive association between them in different tissues, which is possibly influenced by factors specially combined with ZNF133 or the unique upstream regulators on their genes from certain conditions. In our studies, we have revealed the repressive effect of ZNF133/KAP1 complex on L1CAM expression in breast cancer, in which ZNF133 is gradually lost in breast cancer and L1CAM plays an important role in breast tumorigenesis.

Additionally, as we known, there exists intricate crosstalk amongst the components within neurodevelopment and cancer metastasis. For example, whole-genome sequencing of gastric cancer shows that the axon guidance pathway (netrins, ephrins, semaphorins and slit-Robo) is mutated in 59% of the gastric tumors, demonstrating that axon guidance pathway may be involved in the progression of cancer and might be a promising therapeutic target(32, 33). Netrins are firstly identified as axon guidance molecules that guide nerve cells and axons migration during neural development. Increasing evidence has addressed the important role of Netrin-1 in non-neural system, including various types of cancers(34–36). Thus, it is reasonable to reinforce the role of L1CAM, the nervous system protein, in breast cancer metastasis, and approves the involvement of ZNF133 in target L1CAM to suppress progression of breast cancer.

The KAP repressive complex consists of HDAC1, HDAC2, HP1, RBBP4, RBBP7, and SetDB1(37). KAP1 acts as an interaction partner of members of the family of KRAB domain-containing zinc-finger transcription factors, which is also supported by our current study that KRAB domain is involved in ZNF133. Actions of KAP1 depend on its recruitment to chromatin by various transcription factors capable of DNA binding. Thus, recruitment of KAP1, via its RBCC domain, by different transcription factors could elicit different cellular outputs, a possible explanation of the discrepancy of the effects of KAP1 on EMT and survival observed in our experiments versus that reported by others. We show that ZNF133/KAP1 complex targets the transcription of a cohort of genes and regulates several cellular key biological processes including dendrite, perikaryon, and cell proliferation. Clearly, the multitude of the cellular function of the ZNF133 is beyond what we investigated in the current study. However, we are by no means to exclude the involvement and the importance of other downstream target genes, besides L1CAM. Nevertheless, our results indicate that ZNF133 is a transcription repressor and a potential suppressor of L1CAM, adding to the understanding of L1CAM-related breast carcinogenesis and supporting the pursuit of ZNF133 as a novel focus point for breast cancer.

Traditional histopathological classification aims to categorize breast cancer into subgroups to inform clinical management decisions, but the diversity within these subgroups remains considerable. The complexity pervades breast tumors at every level-from (epi)genomic, transcriptomic and proteomic landscapes, through to cellular composition and clinical behavior. This poses great challenges in predicting the development of primary tumors, and treating metastatic disease. We showed that ZNF133 worked as a novel effective biomarker to reflect a good prognosis with less metastasis in breast cancer patients. The expression of ZNF133 was lower in invasive breast carcinoma tissues subtypes, and negatively correlated with advanced pathological grade. Moreover, ZNF133 was effective against L1CAM expression in breast cancer cells. Here, using murine models of breast cancer, we reported that exogenous ZNF133 could suppress breast cancer growth and metastasis in vivo through inhibiting L1CAM. These data suggest that ZNF133 has the potential to act as not only a good prognosis indicator for breast cancer, but also a focus point for clinical trial testing the therapeutic potential of ZNF133-based gene therapy.

ZFPs Zinc finger proteins

KRAB Krüppel associated box

ZNF133 Zinc-finger protein 133

PPCD posterior polymorphous corneal dystrophy

L1CAM L1 cell adhesion molecular

TMA Tissue microarray

GOBO Outcome for Breast cancer Online

GEPIA Gene Expression Profiling Interactive Analysis

EMT epithelial–mesenchymal transition

FPLC fast protein liquid chromatography

GST glutathione S-transferase

ChIP-seq chromatin immunoprecipitation-based deep sequencing

DAVID Database for Annotation, Visualization and Integrated Discovery

ERK1/2 extracellular signal-related kinase 1/2

NCG NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt

Ethics approval and consent to participate

Animal handling and procedures were approved by the Animal Ethical and Welfare Committee (AEWC) of Tianjin Medical University Cancer Institute and Hospital (NSFC-AE-2022199). Use of the TMAs complied with relevant regulations, and was approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital.

Consent for publication

All the authors read and approved the final version of the manuscript to be published.

Availability of data and materials

Data for Kaplan–Meier survival analysis in breast cancer patients are publicly available online at http://kmplot.com/analysis/index.php?p=background. Data for Gene expression-based Outcome for Breast cancer Online (GOBO) analysis are available from http://co.bmc.lu.se/gobo/gsa.pl. Data for Gene Expression Profiling Interactive Analysis (GEPIA) analysis are available from http://gepia.cancer-pku.cn/. Data for mRNA expression analysis in breast cancer patients are available in the Oncomine database at https://www.oncomine.org/. The access to publicly available cancer TCGA data is provided in UALCAN web resource at http://ualcan.path.uab.edu/index.html. The open reading frame, conserved domains, and chromosomal location of ZNF133 were analyzed using the Uniprot (https://www.uniprot.org/) and NCBI (www.ncbi.nlm.nih.gov) databases. The theoretical molecular weight of ZNF133 was calculated using http://www.detaibio.com/sms2/protein_mw.html tools. The homologous alignment and phylogenetic analysis were performed using the MegAlign with Clustal V method.

Competing interests

The authors declare no competing interests.

Funding

This work was supported by grants from the National Natural Science Foundation of China (81902811, 82002977, 82103387 and 82203471), and Tianjin Natural Science Foundation (No. 20JCZDJC00030 and 19JCZDJC65800).

Authors' contributions

LL, XW, and KH conceived and designed the study, performed experiments, and analyzed data; KH and YC performed animal experiments and pathologic analysis; XL analyzed the clinical datasets; LQ, BW, ZW, HW, JH, LH, MW, and ZS provided technical assistance. XL, CB, YC, RC, ML, and CX provided funding support and some ideas. LL drafted the paper; BL and LL discussed and finalized the paper.

Acknowledgements

Not applicable

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ZNF133SupplementaryFigures20221111.pptx

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ZNF133 is a potent suppressor in breast carcinogenesis through dampening L1CAM, a driver for tumor cell invasion

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Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

Tissue microarray (TMA) and immunohistochemistry

Gene Construction And Lentivirus Production

Cell Culture And Transfection

Reagents And Antibodies

Fluorescence Confocal Microscopy

Luciferase Reporter Assay

Immunopurification And Mass Spectrometry

Co-immunoprecipitation And Western Blotting

Gst Pull-down Assay

Fast Protein Liquid Chromatography

Chip Sequencing

Qchip

Quantitative Rt-pcr Analysis

Proliferation Assay

Colony Formation Assay

Cell Invasion Assay

Statistical analysis

Results

A lower expression level of ZNF133 predicts a poorer prognosis in patients with breast cancer

Znf133 Inhibits Proliferation And Invasion Of Breast Cancer Cells

Znf133 Is A Transcription Repressor

Znf133 Is Physically Associated With The Kap1 Complex

Identification Of Genome-wide Transcriptional Targets For The Znf133

Transcription Repression Of L1cam By Znf133/kap1 Complex

Role Of L1cam In Promoting Breast Cancer Progression

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

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