Research ArticleExperimental Studies

Knockdown of CARD14 Inhibits Cell Proliferation and Migration in Breast Cancer Cells

JI-YEON LIM, SEOK WON KIM, BYEOL KIM and SEON-JOO PARK
Anticancer Research April 2020, 40 (4) 1953-1962; DOI: https://doi.org/10.21873/anticanres.14150

Abstract

Background/Aim: Caspase recruitment domain family, member 14 (CARD14) is a member of the CARD family of proteins, which play an important role in immune and inflammatory response, and cell survival and proliferation. Here, we identified the role of CARD14 in human breast cancer. Materials and Methods: Immunohistochemistry was performed to evaluate CARD14 expression in breast cancer. Using CARD14 knockdown cells by small interfering RNA, colony formation and MTT assays, flow cytometry analyses, and migration assays were performed to evaluate the proliferation, cell cycle distribution, apoptosis, and migration ability of MCF7 and SK-BR-3 cells. Results: CARD14 expression was significantly higher in breast cancer samples than in normal breast samples. CARD14 knockdown inhibited cell proliferation and migration, caused cell cycle arrest at the G1/S boundary, and promoted apoptosis. Conclusion: CARD14 regulates the proliferation and migration of MCF7 and SK-BR-3 cells; it is thus, a novel potential therapeutic target in breast cancer.

The proliferating cancer cells can cause a change in the surrounding microenvironment to facilitate their survival, proliferation and metastasis by creating de novo vasculature for oxygen supply to the tumor, perturbation of signal transduction and metabolism in the surrounding stroma and protecting cancer cells from immune cells (1). Emerging evidence suggests that the inflammatory microenvironment plays an important role in tumor development, including its initiation, progression, metastasis and response to therapy (2-4). Especially, NF-ĸB is a major transcription factor involved in the regulation of immune response and inflammatory reactions, and aberrant NF-ĸB activity in TME plays an important role in cancer development and progression (5, 6). Thus, the understanding of NF-ĸB pathway regulation is essential for the therapeutic strategy to overcome cancer.

Caspase recruitment domain (CARD)-containing proteins have been established as scaffolding molecules that regulate immune response, inflammation and tissue homeostasis. CARD family proteins were identified as mediators of activation of the transcription factor NF-ĸB that regulates genes involved in immune and inflammatory response, cell survival, proliferation and cellular stress responses (7, 8). CARD family proteins have a CARD domain at their N-terminus, followed by a coiled-coil (CC) domain, a linker region and a membrane-associated guanylate kinase-like domain (MAGUK), which contains a PDZ, a SH3 and a GUK domain in the C-terminus and are named CARD11 (CARMA1 and Bimp3), CARD14 (CARMA2 and Bimp2) and CARD10 (CARMA3, Bimp1) (9-12). These proteins have been shown to interact with the CARD domain of B-cell lymphoma (BCL10), a protein that acts as a positive regulator of cell apoptosis and NF-ĸB activation and interacts with protease mucosa associated lymphoid tissue lymphoma translocation gene 1 (MALT1). The CARMA-BCL10-MALT1 (CBM) complex induces recruitment of downstream effectors and activation of the NF-ĸB pathway (13, 14). CARD family proteins with conserved domains are known to perform similar functions under different stimuli, but exhibit distinct expression in different tissues. CARD11 is expressed in hematopoietic cells and is essential for antigen receptor-induced NF-ĸB activation in B and T lymphocytes, while CARD10 activates NF-ĸB signaling induced by G-protein-coupled receptors (GPCRs) in various non-hematopoietic cells (15). CARD14 is expressed in the placenta, skin and several mucosal tissues. In addition, CARD family proteins have recently been reported to be expressed in the colonic tissue and are involved in inflammatory activation in patients with inflammatory bowel disease (16). Recently, several studies have reported that the CARD family of proteins plays an important role in the pathogenesis of several types of cancer (17-23).

CARD14 is strongly expressed in the epidermal keratinocyte of the skin and was recently reported to be involved in inflammatory disorders of the human skin, such as psoriasis (24, 25). Numerous sequence variations and mutations in CARD14 are responsible for psoriasis and psoriasis-related diseases, and also a mutation in CARD14 has been reported to lead to enhanced NF-ĸB activation and upregulation of inflammatory transcripts in keratinocytes, such as CXCL8, CCL20, IL8 and IL6 (26-28). Furthermore, three transcript variants of CARD14 have been shown to be produced after alternative splicing. The full-length of CARD14 (CARD14fl) is the longest protein consisting of 1,004 amino acids, a shorter isoform of CARD14 (CARD14sh) lacks the C-terminal SH3 and MAGUK domains, and a CARD14cardless (CARD12cl) lacks the CARD domain and contains a portion of the CC domain and a linker region (29). CARD14sh is still able to activate NF-ĸB signaling similarly to CARD14fl and is predominantly expressed in human skin. Importantly, CARD14 isoforms are expressed in several hematopoietic cells and tissues such as bronchus, cervix, colon and lung as well as cancer cell lines derived from these tissues (26, 29). Although the function of CARD14 in keratinocytes and skin disorders has been investigated, the role of CARD in cancers has not been explored.

In the present study, we aimed to identify the role of CARD14 in breast cancer progression. We found that CARD14fl showed abundant expression in breast cancer cell lines, MCF7 and SK-BR-3. Further experiments to identify the functional role of CARD14 inn breast cancer progression revealed that knockdown of CARD14fl led to decreased breast cancer cell proliferation and migration ability, accompanied by the induction of cell death through NF-ĸB. These results suggest that CARD14 may act as a new therapeutic target in breast cancer treatment strategies.

Materials and Methods

Tissue microarray (TMA). CARD14 expression in breast tumor tissues was assessed by immunohistochemistry using human tissue arrays (CBA; Super Bio Chips; Seoul, Korea). Tissue slide was deparaffinized with xylene, rehydrated with gradient ethanol, and then boiled in 10 mM citrate buffer containing 0.05% Tween-20 for 3 min. Endogenous peroxidase activity was inhibited with 3% H2O2 in 1xPBS for 15 min. The slides were blocked with 5% bovine serum albumin (BSA) in PBS for 1h, and then incubated with anti-CARD14 antibody (Origene, Rockville, MD, USA). After washing with 1xPBS for 15 min, the slides were incubated with HRP-conjugated secondary antibody and detected with DAB solution. Evaluation of CARD14 expression was performed by two pathologists according to the intensity of cellular staining of positively immunoreactive cells. The staining intensity was scored as: 0 (no staining score), 1 (weak staining score), 2 (moderate staining score), and 3 (strong staining score).

Cell culture. Human breast cancer cell lines BT-549, Hs578T, MCF7, MDA-MB-231, MDA-MB-436, SK-BR-3 and human breast epithelial cell line MCF10A were purchased form the American Type Culture Collection (ATCC; Manassas, VA, USA). MCF10A cells were cultured in Mammary Epithelial Cell Growth Medium (MEGM, Lonza) and Hs578T, MDA-MB-436 were cultured in DMEM containing 10% feral bovine serum (FBS). Other cells were cultured in RPMI1640 containing 10% FBS. All breast cancer cell lines were cultured at 37°C, in humidified air containing 5% CO2.

Small-interference RNA (siRNA) transfection. MCF7 and SK-BR-3 cells were transfected with siRNA oligonucleotide duplexes against human CARD14 and a scrambled control siRNA using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The siRNA sequences targeting CARD14 (CARD14 siRNA #1; 5’-GCCAGGUUCUAGAGGAUGA-3’ CARD14 siRNA #2; 5’-GGACAAGUCUCUCAGCUAU-3’) were designed and synthesized for transient transfection. AccuTarget Control siRNA (Bioneer; Dajeon, Korea) was used as a negative control. Two days after transfection, the efficiency of CARD14 knockdown was confirmed by measuring mRNA and protein expression levels.

Quantitative real time-PCR (qRT-PCR). The RNA was extracted using Trizol reagent (Invitrogen), and cDNA synthesis was performed by Primescript™ Reverse Transcriptase (TaKaRa Bio Inc., Shiga, Japan). qRT-PCR was carried out using SYBR Premix Ex Taq kit (TaKaRa Bio Inc.) and a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The sequences of the target genes specific primers were as follows: CARD14 forward 5’-GCCTGGCTCCTGA CGG-3’, reverse 5’-TCGGGGGCTCTGCTC C-3’; human GAPDH forward 5’-TCAAGAAGGTGGTGAAGCAG-3’, reverse 5’-TCCACCACCCTGTTGCTGT A-3’. CARD14 mRNA expression levels were analyzed with the CFX Maestro software (Bio-Rad), and the relative expression of mRNA was calculated by the 2−ΔΔCt method. GAPDH was used to normalize gene expression levels.

Western blot analysis. Cells were harvested, washed twice with PBS, and then lysed with RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 10 μg/ml leupeptin and 10 μg/ml aprotinin) for 30 min on ice. Lysates were collected by centrifugation at 15,000 × g for 15 min, and then equal amounts of lysates were electrophoresed on SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). The membranes were probed with anti-CARD14 antibody and anti-β-actin (Santa Cruz, CA, USA) overnight at 4°C, and then incubated with peroxidase-conjugated secondary antibody. Protein bands were detected by the ECL solution.

Cell proliferation assay. Cell proliferation was analysed by the MTT assay. A total of 1×103 cells were seeded in 96-well plates. After 1, 2, 3 and 4 days of culture, the MTT reagent was added to the cells at a concentration of 5 mg/ml for 4 h at 37°C. The cultured media were removed and the cells were dissolved with 0.04 N HCL in isopropanol. The absorbance of each well was measured at 570 nm using the Epoch Microplate Spectrophotometer (BioTek Instruments Inc., Winoski, VT, USA).

Figure 1.
Figure 1.

Expression of CARD14 in human breast cancer tissues and cell lines. (A) Expression levels of CARD14 protein in breast cancer tissues by TMA analysis. (B) Expression of CARD14fl mRNA expression level was examined in breast cancer cell lines BT-549, Hs578T, MCF7, MDA-MB-231, SK-BR-3 and human breast epithelial cell line MCF10A using quantitative real-time PCR. *p<0.05, indicated significant difference compared to normal. (C) CARD14fl protein expression was also examined using western blot analysis. β-actin was used as a loading control. **p<0.01, indicated a significant difference compared to MCF10A.

Colony formation assay. Negative control and CARD14 siRNA transfected cells were grown in 60 mm cell culture dishes at a density of 500 cells/dish for 14 days. Colonies were washed twice with PBS, fixed with ice-cold 95% methanol, and stained with methylene blue for 5 min at room temperature. Colonies were washed with tap water and images from 4 random fields were taken using an inverted microscope.

Cell cycle analysis. Two days after the CARD14 siRNAs were transfected into the cells, cell cycle distribution was analysed by flow cytometry. The cells were trypsinized and fixed in ice-cold 70% ethanol, and then stained with propidium iodide (PI; 50 μg/ml) in the presence of RNase A (0.05 μg/ml) for 15 min in the dark. Samples were analysed using a flow cytometer (FACS; BD biosciences, San Jose, CA, USA) and the ModFit LT™ software (Verity Software House, Topsham, ME, USA). For apoptotic cell death, sub-G0/G1 populations were measured by flow cytometry after staining with propidium iodide (PI) and analyzed with the ModFit LT™ software.

Cell migration assay. Cell migration assay was performed using a 24-well Transwell chamber with 8.0 μm Pore Polycarbonate Membrane Insert (Corning USA). A total of 5×104 cells were suspended in 100 μl of serum-free medium and loaded in the upper chamber, which was pre-warmed with the same volume of serum-free medium at 37°C, and 600 μl of complete medium were added to the lower chamber. After incubated for 24 h, the cells remaining at the upper surface of the membrane were removed with cotton swabs, and the transwell inserts were washed twice with PBS. The migrated cells to the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. The migrated cells were photographed by using an inverted microscope.

Figure 2.
Figure 2.

Knockdown of CARD14 with siRNA in the MCF7 and SK-BR-3 cells. (A) mRNA expression levels of CARD14 in control siRNA and CARD14 siRNA #1, #2 cells were measured by qRT-PCR. (B) CARD14 protein expression levels were confirmed by western blot analysis. β-actin was used as the internal control. Data are presented as means±SD of three independent experiments. *p<0.05, **p<0.01, indicated significant differences compared to control siRNA transfected cells.

Figure 3.
Figure 3.

Reduction in cell growth and proliferation by CARD14 knockdown in MCF7 and SK-BR-3 cells. (A) Cell proliferation of CARD14 knockdown cells was analyzed at 1, 2, 3 and 4 days by the MTT assay. Cells were CARD14 was silenced showed significantly reduced viability compared with control cells. (B) Cell proliferation in cells transfected with CARD14 siRNA was assessed using colony formation assay. *p<0.05, ***p<0.005, indicated significant differences compared to control siRNA.

Statistical analysis. Data from all experiments are represented as mean±SD. Statistical comparisons were carried out using student's t-test. We considered p<0.05(**) as significant. Analyses were carried out with Prism software (GraphPad) and Excel (Microsoft).

Results

CARD14 was expressed in human breast cancer tissues and cell lines. Tissue microarray (TMA) analysis was used to identify the correlation between CARD14 expression and breast cancer. The use of tissue microarrays that contained normal tissues found adjacent to the cancer (n=10) and breast cancer tissues (n=50) also showed increased expression of CARD14 in breast cancer tissues (Figure 1A). We next assessed the expression of CARD14 in several breast cancer cell lines. qRT-PCR was performed in BT-549, Hs578T, MCF7, MDA-MB-231, MDA-MB-436, SK-BR-3 and human breast epithelial cell line MCF10A. As shown in Figure 1B, CARD14fl mRNA expression was significantly elevated in MCF7 and SK-BR-3 cells compared to MCF10A. Next, CARD14fl protein expression levels in these cells were assessed by western blot analysis using an antibody against the full length of human CARD14. As shown in Figure 1C, CARD14fl protein was strongly expressed in MCF7 and SK-BR-3 cells.

Figure 4.
Figure 4.

CARD14 knockdown induced cell cycle arrest in MCF7 and SK-BR-3 cells. (A) Control and CARD14 siRNA transfected cells were subjected to cell cycle analysis by FACS. (B) Cell cycle distributions were analyzed by Modfit LT software and numbers of cells in each phase of the cell cycle are represented by bar graphs. CARD14 knockdown led to arrest at the G0/G1 phase of the cell cycle. *p<0.05, **p<0.01 and ***p<0.005, indicated significant differences compared to control siRNA.

Figure 5.
Figure 5.

Induction of apoptosis in CARD14 knockdown MCF7 and SK-BR-3 cells. Sub-G0/G1 phase cells in the cells treated with control and CARD14 siRNAs were analysed by flow cytometry after PI staining. Bar graph represents the percentage of cells undergoing apoptosis. Data are presented as mean±SD of three independent experiments. *p<0.05, **p<0.01, indicated significant differences compared to control siRNA.

Figure 6.
Figure 6.

Effect of CARD14 knockdown on the cell migration of MCF7 and SK-BR-3 cells. (A, B). The cells transfected with control siRNA and CARD14 siRNAs were analysed by transwell migration assay. The images were obtained from four randomly chosen fields and the number of migrated cells is shown by a bar graph. Data are presented as mean±SD of three independent experiments. **p<0.01, ***p<0.005, indicated significant differences compared to control siRNA.

Knockdown of CARD14fl through specific siRNA. To identify the role of CARD14 in breast cancer cell proliferation, we used two siRNAs to silence the expression of CARD14fl. MCF7 and SK-BR-3 cells were transfected with CARD14 siRNA #1 and #2 and negative control siRNA. After 2 days, the knockdown efficiency of CARD14 was evaluated by qRT-PCR. As shown in Figure 2A, CARD14 mRNA expression was significantly reduced in CARD14 siRNA #1 and #2 transfected MCF7 and SK-BR-3 cells compared to control siRNA transfected cells. Furthermore, western blot analysis also confirmed that CARD14 protein expression was reduced by more than 90% in CARD14 knockdown cells compared to control cells (Figure 2B).

CARD14 knockdown reduced cell viability and proliferation in MCF7 and SK-BR-3 cells. We measured cell viability by the MTT assay to confirm the effect of CARD14 knockdown on cell growth. MCF7 and SK-BR-3 cells were transfected with CARD14 siRNA #1 and #2, and then subjected to MTT assay at 1, 2, 3 and 4 days. As shown in Figure 3A, cell viability was markedly inhibited in CARD14 siRNA #1 and #2- transfected MCF7 and SK-BR-3 cells compared with control siRNA-transfected cells. Next, colony formation analysis was additionally performed to identify the clonogenic ability of CARD14. The number of colonies in CARD14-depleted MCF7 and SK-BR-3 cells was reduced compared to the control cells (Figure 3B). These results indicated that knockdown of CARD14 significantly suppressed cell proliferation and inhibited colony formation of breast cancer cells.

CARD14 knockdown induces cell-cycle arrest and apoptosis in MCF7 and SK-BR-3 cells. Since aberrant cell proliferation is associated with deregulation of the cell cycle (30), we analysed the cell cycle of CARD14 knockdown MCF7 cells as well as SK-BR-3 cells using flow cytometry. As shown in Figure 4A, B, CARD14 siRNA #1 and #2 MCF7 and SK-BR-3 cells showed accumulation in the G0/G1 phase of the cell cycle compared with control siRNA treated cells. Moreover, knockdown of CARD14 decreased the percentage of cells in the S phase. These results demonstrated that knockdown of CARD14 led to cell cycle arrest at the G0/G1 phase, resulting in cell growth inhibition. In addition, overexpression of CARD14 has been reported to play a protective role against apoptosis (29). The slightly increased percentage of cells at the sub-G0/G1 phase indicates apoptotic cell death in CARD14 knockdown MCF7 (Figure 5A) and SK-BR-3 cells (Figure 5B). These results suggest that knockdown of CARD14 may induce cell apoptosis.

CARD14 knockdown decreased cell migration of MCF7 and SK-BR-3 cells. In order to determine whether CARD14 is involved in the tumorigenic capacity of breast cancer cells, we conducted a transwell migration assay. As shown in Figure 6A, the number of migrated cells was significantly reduced in CARD14 knockdown MCF7 cells. Similarly, CARD14 siRNA-transfected SK-BR-3 cells exhibited a decrease in cell migration compared with control siRNA-transfected cells (Figure 6B). Therefore, CARD14 may be involved in the cell migration of breast adenocarcinoma cells.

Discussion

CARD14 is one of the members of the CARD family of proteins, which play an important role in the regulation of cell death, cancer cell proliferation, and resistance to chemotherapy (31, 32). CARD family proteins are classified according to a tissue-specific expression pattern despite their conservative structure and function (9). Although CARD family proteins have been established as key regulators of immune response and cytokine production, several CARD proteins are involved in tumorigenesis. Unlike other CARD family proteins, previous studies have confirmed the existence of CARD14 isoforms (CARD14fl, CARD14sh and CRAD14cl) and mainly studied the biological role of CARD14sh because this is the most abundantly expressed isoform. Furthermore, the mRNA expression of CARD14 isoforms was detected in several hematopoietic cells and tissues such as the bronchus, cervix, colon and lung as well as in epithelial cell lines derived from these tissues (26, 27, 29). These results suggest the possibility that CARD14 has functions in various tissues besides the skin. However, its biological roles in other cancers have not been studied. In this study, we found that the full-length CARD14 protein was not only expressed in the breast cancer cell lines MCF7 and SK-BR-3, but also in the lung cancer cell lines H358 and CALU-3, in the colorectal cancer cell line DLD-1 and in the cervical cancer cell line HeLa (data not shown). In particular, we focused on identifying the role of CARD14 in breast cancer. TCGA data analysis, revealed that CARD14 mRNA is highly expressed in breast tumor tissues. In addition, immunohistochemistry with a specific antibody against CARD14 also showed its high expression in breast cancer tissues compared to normal ones. We next determined the expression patterns of CARD14 in breast cancer cell lines, MCF7 and SK-BR-3, which have high expression levels of CARD14fl, in order to identify the functional role of CARD14 in tumorigenesis. To explore the biological role of CARD14 in breast cancer cell lines, we inhibited the expression of CARD14 in MCF7 and SK-BR-3 cells by siRNA. Western blot analysis revealed that CARD14 expression in both cell lines was successfully suppressed by siRNA specific for CARD14. Knockdown of CARD14 resulted in a significant decrease in cell viability, proliferation and colony formation in both cell lines. In most cells, the capacity of proliferation is conferred by cell growth factors that regulate cell cycle progression. For this reason, we conducted a cell cycle analysis in MCF7 and SK-BR3 cells transfected with or without tCARD14 siRNA. Knockdown of CARD14 induced cell cycle arrest at the G0/G1 phase. We also examined whether CARD14 is involved in tumorigenesis. Soft-agar assay and migration assay showed that knockdown of CARD14 led to a decrease in anchorage-independent cell proliferation and the migration ability of MCF7 and SK-BR-3 cells. These results suggested that CARD14fl regulates breast cancer cell proliferation, and apoptosis and induces migration.

Interestingly, although CARD14 is less characterized compared to other CARD family proteins, several studies have reported the structure and expression pattern of CARD14 isoforms. Three types of CARD14 isoforms (CARD14fl, CARD14sh and CARD14cl), which are produced by alternative splicing, are differentially expressed in epithelial cells from diverse tissues, suggesting distinct intracellular mechanisms in different cell types. The subcellular localization of CARD14 isoforms depends on their structural differences. CARD14fl, which contains domains essential for membrane localization triggers intracellular signaling by stimuli through cell membranes. Although CARD14sh is the most abundantly expressed, whereas CARD14cl is expressed in few cell types, CARD14sh and CARD14cl both have a cytoplasmic localization (9, 29). When CARD14fl is overexpressed by membrane receptor activation in response to stimulation, it can be associated with BCL10 and MALT1 to form a CBM complex, resulting in promotion of NF-ĸB activation.

However, CARD14sh and CARD14cl have been shown to regulate the NF-ĸB signal transduction pathway by acting in intracellular organelles. It has been reported that CARD14sh interacts with TRAF2, which is involved in the activation of the NF-ĸB pathway in response to ER stress (33, 34). The shortest CARD14 isoform, CARD14cl, acts as a negative regulator, and may partially protect from apoptosis in a NF-ĸB-independent manner, suggesting the existence of other protective mechanisms (29). Therefore, further studies are needed to confirm the expression pattern of CARD14 isoforms and their potential functions in breast cancer cell lines.

In conclusion, this study found that CARD14 is overexpressed in breast cancer tissues and cell lines. CARD14fl is involved in the regulation of proliferation of breast cancer cells. CARD14 may be used as a novel drug target for new cancer treatments.

Acknowledgements

This work is supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Education [NRF-2015R1A5A2009070, NRF-2018R1D1A 1B07048069].

Footnotes

  • ↵* These Authors contributed equally to this work.

  • Author's Contributions

    Seok Won Kim and Seon-Joo Park designed the study, analyzed the result and prepared the manuscripts. Ji-Yeon Lim and Byeol Kim performed experiment, collected data and prepared the figures. All Authors participated in the writing and revision of the final manuscript.

  • Conflicts of Interest

    The Authors declare no conflicts of interest regarding this study.

  • Received February 17, 2020.
  • Revision received February 24, 2020.
  • Accepted February 25, 2020.

References

  1. ↵
    1. Wang M,
    2. Zhao J,
    3. Zhang L,
    4. Wei F,
    5. Lian Y,
    6. Wu Y,
    7. Gong Z,
    8. Zhang S,
    9. Zhou J,
    10. Cao K,
    11. Li X,
    12. Xiong W,
    13. Li G,
    14. Zeng Z,
    15. Guo C
    : Role of tumor microenvironment in tumorigenesis. J Cancer 8(5): 761-773, 2017. PMID: 28382138. DOI: 10.7150/jca.17648
    OpenUrlCrossRefPubMed
  2. ↵
    1. de Visser KE,
    2. Coussens LM
    : The inflammatory tumor microenvironment and its impact on cancer development. Contrib Microbiol 13: 118-137, 2006. PMID: 16627962. DOI: 10.1159/000092969
    OpenUrlCrossRefPubMed
    1. Spill F,
    2. Reynolds DS,
    3. Kamm RD,
    4. Zaman MH
    : Impact of the physical microenvironment on tumor progression and metastasis. Curr Opin Biotechnol 40: 41-48, 2016. PMID: 26938687. DOI: 10.1016/j.copbio.2016.02.007
    OpenUrlCrossRefPubMed
  4. ↵
    1. Joyce JA,
    2. Fearon DT
    : T cell exclusion, immune privilege, and the tumor microenvironment. Science 348(6230): 74-80, 2015. PMID: 25838376. DOI: 10.1126/science.aaa6204
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Bao B,
    2. Thakur A,
    3. Li Y,
    4. Ahmad A,
    5. Azmi AS,
    6. Banerjee S,
    7. Kong D,
    8. Ali S,
    9. Lum LG,
    10. Sarkar FH
    : The immunological contribution of NF-kB within the tumor microenvironment: A potential protective role of zinc as an anti-tumor agent. Biochim Biophys Acta 1825(2): 160-172, 2012. PMID: 22155217. DOI: 10.1016/j.bbcan.2011.11.002
    OpenUrlPubMed
  6. ↵
    1. Xia Y,
    2. Shen S,
    3. Verma IM
    : NF-ĸB, an active player in human cancers. Cancer Immunol Res 2(9): 823-830, 2014. PMID: 25187272, DOI: 10.1158/2326-6066.CIR-14-0112
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Jiang C,
    2. Lin X
    : Regulation of nf-kappab by the card proteins. Immunol Rev 246(1): 141-153, 2012. PMID: 22435552. DOI: 10.1111/j.1600-065X.2012.01110.x
    OpenUrlCrossRefPubMed
  8. ↵
    1. Blonska M,
    2. Lin X
    : NF-kappaB signaling pathways regulated by carma family of scaffold proteins. Cell Res 21(1): 55-70, 2011. PMID: 21187856. DOI: 10.1038/cr.2010.182
    OpenUrlCrossRefPubMed
  9. ↵
    1. Scudiero I,
    2. Vito P,
    3. Stilo R
    : The three carma sisters: So different, so similar: A portrait of the three carma proteins and their involvement in human disorders. J Cell Physiol 229(8): 990-997, 2014. PMID: 24375035. DOI: 10.1002/jcp.24543
    OpenUrlCrossRefPubMed
    1. Bertin J,
    2. Wang L,
    3. Guo Y,
    4. Jacobson MD,
    5. Poyet JL,
    6. Srinivasula SM,
    7. Merriam S,
    8. DiStefano PS,
    9. Alnemri ES
    : CARD11 and CARD14 are novel caspase recruitment domain (card)/membrane-associated guanylate kinase (maguk) family members that interact with bcl10 and activate NF-kappa b. J Biol Chem 276(15): 11877-11882, 2001. PMID: 11278692. DOI: 10.1074/jbc.M010512200
    OpenUrlAbstract/FREE Full Text
    1. Gaide O,
    2. Martinon F,
    3. Micheau O,
    4. Bonnet D,
    5. Thome M,
    6. Tschopp J
    : Carma1, a card-containing binding partner of bcl10, induces bcl10 phosphorylation and NF-kappab activation. FEBS Lett 496(2-3): 121-127, 2001. PMID: 11356195. DOI: 10.1016/s0014-5793(01)02414-0
    OpenUrlCrossRefPubMed
  12. ↵
    1. McAllister-Lucas LM,
    2. Inohara N,
    3. Lucas PC,
    4. Ruland J,
    5. Benito A,
    6. Li Q,
    7. Chen S,
    8. Chen FF,
    9. Yamaoka S,
    10. Verma IM,
    11. Mak TW,
    12. Nunez G
    : Bimp1, a maguk family member linking protein kinase c activation to bcl10-mediated nf-kappab induction. J Biol Chem 276(33): 30589-30597, 2001. PMID: 11387339. DOI: 10.1074/jbc.M103824200
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Willis TG,
    2. Jadayel DM,
    3. Du MQ,
    4. Peng H,
    5. Perry AR,
    6. Abdul-Rauf M,
    7. Price H,
    8. Karran L,
    9. Majekodunmi O,
    10. Wlodarska I,
    11. Pan L,
    12. Crook T,
    13. Hamoudi R,
    14. Isaacson PG,
    15. Dyer MJ
    : Bcl10 is involved in t(1;14)(p22;q32) of malt b cell lymphoma and mutated in multiple tumor types. Cell 96(1): 35-45, 1999. PMID: 9989495. DOI: 10.1016/s0092-8674(00)80957-5
    OpenUrlCrossRefPubMed
  14. ↵
    1. Zhang S,
    2. Lin X
    : Carma3: Scaffold protein involved in nf-kappab signaling. Front Immunol 10: 176, 2019. PMID: 30814996. DOI: 10.3389/fimmu.2019.00176
    OpenUrlCrossRefPubMed
  15. ↵
    1. McAuley JR,
    2. Freeman TJ,
    3. Ekambaram P,
    4. Lucas PC,
    5. McAllister-Lucas LM
    : Carma3 is a critical mediator of g protein-coupled receptor and receptor tyrosine kinase-driven solid tumor pathogenesis. Front Immunol 9: 1887, 2018. PMID: 30158935. DOI: 10.3389/fimmu.2018.01887
    OpenUrlPubMed
  16. ↵
    1. Yamamoto-Furusho JK,
    2. Fonseca-Camarillo G,
    3. Furuzawa-Carballeda J,
    4. Sarmiento-Aguilar A,
    5. Barreto-Zuniga R,
    6. Martinez-Benitez B,
    7. Lara-Velazquez MA
    : Caspase recruitment domain (card) family (card9, card10, card11, card14 and card15) are increased during active inflammation in patients with inflammatory bowel disease. J Inflamm (Lond) 15: 13, 2018. PMID: 30008619. DOI: 10.1186/s12950-018-0189-4
    OpenUrlPubMed
  17. ↵
    1. Liu X,
    2. Zhang X,
    3. Bi J,
    4. Li Z,
    5. Zhang Z,
    6. Kong C
    : Caspase recruitment domain family member 10 regulates carbamoyl phosphate synthase 1 and promotes cancer growth in bladder cancer cells. J Cell Mol Med 23(12): 8128-8138, 2019. PMID: 31565867. DOI: 10.1111/jcmm.14683
    OpenUrlPubMed
    1. Xia ZX,
    2. Li ZX,
    3. Zhang M,
    4. Sun LM,
    5. Zhang QF,
    6. Qiu XS
    : Carma3 regulates the invasion, migration, and apoptosis of non-small cell lung cancer cells by activating nf-small ka, cyrillicb and suppressing the p38 mapk signaling pathway. Exp Mol Pathol 100(2): 353-360, 2016. PMID: 26526492. DOI: 10.1016/j.yexmp.2015.10.004
    OpenUrlPubMed
    1. Du S,
    2. Jia L,
    3. Zhang Y,
    4. Fang L,
    5. Zhang X,
    6. Fan Y
    : Carma3 is upregulated in human pancreatic carcinoma, and its depletion inhibits tumor proliferation, migration, and invasion. Tumour Biol 35(6): 5965-5970, 2014. PMID: 24633921. DOI: 10.1007/s13277-014-1791-6
    OpenUrl
    1. Zhao T,
    2. Miao Z,
    3. Wang Z,
    4. Xu Y,
    5. Wu J,
    6. Liu X,
    7. You Y,
    8. Li J
    : Carma3 overexpression accelerates cell proliferation and inhibits paclitaxel-induced apoptosis through NF-kappab regulation in breast cancer cells. Tumour Biol 34(5): 3041-3047, 2013. PMID: 23708960. DOI: 10.1007/s13277-013-0869-x
    OpenUrl
    1. Miao Z,
    2. Zhao T,
    3. Wang Z,
    4. Xu Y,
    5. Song Y,
    6. Wu J,
    7. Xu H
    : Carma3 is overexpressed in colon cancer and regulates NF-kappab activity and cyclin D1 expression. Biochem Biophys Res Commun 425(4): 781-787, 2012. PMID: 22884800. DOI: 10.1016/j.bbrc.2012.07.152
    OpenUrlPubMed
    1. Watt SA,
    2. Purdie KJ,
    3. den Breems NY,
    4. Dimon M,
    5. Arron ST,
    6. McHugh AT,
    7. Xue DJ,
    8. Dayal JH,
    9. Proby CM,
    10. Harwood CA,
    11. Leigh IM,
    12. South AP
    : Novel card11 mutations in human cutaneous squamous cell carcinoma lead to aberrant NF-KappaB regulation. Am J Pathol 185(9): 2354-2363, 2015. PMID: 26212909. DOI: 10.1016/j.ajpath.2015.05.018
    OpenUrlCrossRefPubMed
  23. ↵
    1. Gu L,
    2. Sun W
    : Mir-539 inhibits thyroid cancer cell migration and invasion by directly targeting carma1. Biochem Biophys Res Commun 464(4): 1128-1133, 2015. PMID: 26206083. DOI: 10.1016/j.bbrc.2015.07.090
    OpenUrlPubMed
  24. ↵
    1. Zotti T,
    2. Polvere I,
    3. Voccola S,
    4. Vito P,
    5. Stilo R
    : CARD14/CARMA2 signaling and its role in inflammatory skin disorders. Front Immunol 9: 2167, 2018. PMID: 30319628. DOI: 10.3389/fimmu.2018.02167
    OpenUrlCrossRefPubMed
  25. ↵
    1. Harden JL,
    2. Lewis SM,
    3. Pierson KC,
    4. Suarez-Farinas M,
    5. Lentini T,
    6. Ortenzio FS,
    7. Zaba LC,
    8. Goldbach-Mansky R,
    9. Bowcock AM,
    10. Lowes MA
    : CARD14 expression in dermal endothelial cells in psoriasis. PLoS One 9(11): e111255, 2014. PMID: 25369198. DOI: 10.1371/journal.pone.0111255
    OpenUrlCrossRefPubMed
  26. ↵
    1. Jordan CT,
    2. Cao L,
    3. Roberson ED,
    4. Pierson KC,
    5. Yang CF,
    6. Joyce CE,
    7. Ryan C,
    8. Duan S,
    9. Helms CA,
    10. Liu Y,
    11. Chen Y,
    12. McBride AA,
    13. Hwu WL,
    14. Wu JY,
    15. Chen YT,
    16. Menter A,
    17. Goldbach-Mansky R,
    18. Lowes MA,
    19. Bowcock AM
    : Psors2 is due to mutations in CARD14. Am J Hum Genet 90(5): 784-795, 2012. PMID: 22521418. DOI: 10.1016/j.ajhg.2012.03.012
    OpenUrlCrossRefPubMed
  27. ↵
    1. Jordan CT,
    2. Cao L,
    3. Roberson ED,
    4. Duan S,
    5. Helms CA,
    6. Nair RP,
    7. Duffin KC,
    8. Stuart PE,
    9. Goldgar D,
    10. Hayashi G,
    11. Olfson EH,
    12. Feng BJ,
    13. Pullinger CR,
    14. Kane JP,
    15. Wise CA,
    16. Goldbach-Mansky R,
    17. Lowes MA,
    18. Peddle L,
    19. Chandran V,
    20. Liao W,
    21. Rahman P,
    22. Krueger GG,
    23. Gladman D,
    24. Elder JT,
    25. Menter A,
    26. Bowcock AM
    : Rare and common variants in CARD14, encoding an epidermal regulator of nf-kappab, in psoriasis. Am J Hum Genet 90(5): 796-808, 2012. PMID: 22521419. DOI: 10.1016/j.ajhg.2012.03.013
    OpenUrlCrossRefPubMed
  28. ↵
    1. Van Nuffel E,
    2. Schmitt A,
    3. Afonina IS,
    4. Schulze-Osthoff K,
    5. Beyaert R,
    6. Hailfinger S
    : Card14-mediated activation of paracaspase malt1 in keratinocytes: Implications for psoriasis. J Invest Dermatol 137(3): 569-575, 2017. PMID: 27939769. DOI: 10.1016/j.jid.2016.09.031
    OpenUrlPubMed
  29. ↵
    1. Scudiero I,
    2. Zotti T,
    3. Ferravante A,
    4. Vessichelli M,
    5. Vito P,
    6. Stilo R
    : Alternative splicing of carma2/card14 transcripts generates protein variants with differential effect on nf-kappab activation and endoplasmic reticulum stress-induced cell death. J Cell Physiol 226(12): 3121-3131, 2011. PMID: 21302310. DOI: 10.1002/jcp.22667
    OpenUrlCrossRefPubMed
  30. ↵
    1. Gerard C,
    2. Goldbeter A
    : The balance between cell cycle arrest and cell proliferation: Control by the extracellular matrix and by contact inhibition. Interface Focus 4(3): 20130075, 2014. PMID: 24904738. DOI: 10.1098/rsfs.2013.0075
    OpenUrlCrossRefPubMed
  31. ↵
    1. Damiano JS,
    2. Reed JC
    : Card proteins as therapeutic targets in cancer. Curr Drug Targets 5(4): 367-374, 2004. PMID: 15134219. DOI: 10.2174/1389450043345470
    OpenUrlCrossRefPubMed
  32. ↵
    1. Juilland M,
    2. Thome M
    : Holding all the cards: How malt1 controls carma/card-dependent signaling. Front Immunol 9: 1927, 2018. PMID: 30214442. DOI: 10.3389/fimmu.2018.01927
    OpenUrlCrossRefPubMed
  33. ↵
    1. Hu P,
    2. Han Z,
    3. Couvillon AD,
    4. Kaufman RJ,
    5. Exton JH
    : Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through ire1alpha-mediated NF-KappaB activation and down-regulation of TRAF2 expression. Mol Cell Biol 26(8): 3071-3084, 2006. PMID: 16581782. DOI: 10.1128/mcb.26.8.3071-3084.2006
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Mauro C,
    2. Crescenzi E,
    3. De Mattia R,
    4. Pacifico F,
    5. Mellone S,
    6. Salzano S,
    7. de Luca C,
    8. D'Adamio L,
    9. Palumbo G,
    10. Formisano S,
    11. Vito P,
    12. Leonardi A
    : Central role of the scaffold protein tumor necrosis factor receptor-associated factor 2 in regulating endoplasmic reticulum stress-induced apoptosis. J Biol Chem 281(5): 2631-2638, 2006. PMID: 16299380. DOI: 10.1074/jbc.M502181200
    OpenUrlAbstract/FREE Full Text
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Knockdown of CARD14 Inhibits Cell Proliferation and Migration in Breast Cancer Cells
JI-YEON LIM, SEOK WON KIM, BYEOL KIM, SEON-JOO PARK
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