Subunit-Specific Role of NF-κB in Cancer

Kaltschmidt B, Greiner J, Kadhim H, Kaltschmidt C. Subunit-Specific Role of NF-κB in Cancer. Biomedicines. 2018;6(2): 44.The transcription factor NF-kB is a key player in inflammation, cancer development, and progression. NF-kB stimulates cell proliferation, prevents apoptosis, and could promote tumor angiogenesis as well as metastasis. Extending the commonly accepted role of NF-kB in cancer formation and progression, different NF-kB subunits have been shown to be active and of particular importance in distinct types of cancer. Here, we summarize overexpression data of the NF-kB subunits RELA, RELB, and c-REL (referring to the v-REL, which is the oncogene of Reticuloendotheliosis virus strain T) as well as of their upstream kinase inhibitor, namely inhibitor of kB kinases (IKK), in different human cancers, assessed by database mining. These data argue against a universal mechanism of cancer-mediated activation of NF-kB, and suggest a much more elaborated mode of NF-kB regulation, indicating a tumor type-specific upregulation of the NF-kB subunits. We further discuss recent findings showing the diverse roles of NF-kB signaling in cancer development and metastasis in a subunit-specific manner, emphasizing their specific transcriptional activity and the role of autoregulation. While non-canonical NF-kB RELB signaling is described to be mostly present in hematological cancers, solid cancers reveal constitutive canonical NF-kB RELA or c-REL activity. Providing a linkage to cancer therapy, we discuss the recently described pivotal role of NF-kB c-REL in regulating cancer-targeting immune responses. In addition, current strategies and ongoing clinical trials are summarized, which utilize genome editing or drugs to inhibit the NF-kB subunits for cancer treatment

CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle

Slotta C, Schlüter T, Ruiz-Perera LM, et al. CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle. PLOS ONE. 2017;12(8): e0182373.Cervical cancer is the fourth common cancer in women resulting worldwide in 266,000 deaths per year. Belonging to the carcinomas, new insights into cervical cancer biology may also have great implications for finding new treatment strategies for other kinds of epithelial cancers. Although the transcription factor NF-κB is known as a key player in tumor formation, the relevance of its particular subunits is still underestimated. Here, we applied CRISPR/Cas9n-mediated genome editing to successfully knockout the NF-κB subunit c-REL in HeLa Kyoto cells as a model system for cervical cancers. We successfully generated a homozygous deletion in the c-REL gene, which we validated using sequencing, qPCR, immunocytochemistry, western blot analysis, EMSA and analysis of off-target effects. On the functional level, we observed the deletion of c-REL to result in a significantly decreased cell proliferation in comparison to wildtype (wt) without affecting apoptosis. The impaired proliferative behavior of c-REL-/- cells was accompanied by a strongly decreased amount of the H2B protein as well as a significant delay in the prometaphase of mitosis compared to c-REL+/+ HeLa Kyoto cells. c-REL-/- cells further showed significantly decreased expression levels of c-REL target genes in comparison to wt. In accordance to our proliferation data, we observed the c-REL knockout to result in a significantly increased resistance against the chemotherapeutic agents 5-Fluoro-2’-deoxyuridine (5-FUDR) and cisplatin. In summary, our findings emphasize the importance of c-REL signaling in a cellular model of cervical cancer with direct clinical implications for the development of new treatment strategies

Subunit-Specific Role of NF-κB in Cancer

The transcription factor NF-κB is a key player in inflammation, cancer development, and progression. NF-κB stimulates cell proliferation, prevents apoptosis, and could promote tumor angiogenesis as well as metastasis. Extending the commonly accepted role of NF-κB in cancer formation and progression, different NF-κB subunits have been shown to be active and of particular importance in distinct types of cancer. Here, we summarize overexpression data of the NF-κB subunits RELA, RELB, and c-REL (referring to the v-REL, which is the oncogene of Reticuloendotheliosis virus strain T) as well as of their upstream kinase inhibitor, namely inhibitor of κB kinases (IKK), in different human cancers, assessed by database mining. These data argue against a universal mechanism of cancer-mediated activation of NF-κB, and suggest a much more elaborated mode of NF-κB regulation, indicating a tumor type-specific upregulation of the NF-κB subunits. We further discuss recent findings showing the diverse roles of NF-κB signaling in cancer development and metastasis in a subunit-specific manner, emphasizing their specific transcriptional activity and the role of autoregulation. While non-canonical NF-κB RELB signaling is described to be mostly present in hematological cancers, solid cancers reveal constitutive canonical NF-κB RELA or c-REL activity. Providing a linkage to cancer therapy, we discuss the recently described pivotal role of NF-κB c-REL in regulating cancer-targeting immune responses. In addition, current strategies and ongoing clinical trials are summarized, which utilize genome editing or drugs to inhibit the NF-κB subunits for cancer treatment

Overexpression of <i>REL</i> in human cancers.

<p>Overexpression of <i>REL</i> in human cancers.</p

Successful validation of the <i>c-REL</i> knockout in HeLa Kyoto cells on DNA, mRNA and protein level.

<p><b>A:</b> Genomic PCR depicting a profound deletion of the <i>c-REL</i> gene in the <i>c-REL</i> knockout clone (band at 300 bp) compared to the wt clone (band at 700 bp). <b>B:</b> Sequencing analysis confirmed the knockout in exon 2 of <i>c-REL</i>. <b>C:</b> qPCR with specific primers in targeted deletion of exon 2 showed no expression of <i>c-REL</i> on mRNA level in the <i>c-REL</i> knockout clone in comparison to wt. <b>D:</b> Western blot analysis validated the knockout of <i>c-REL</i> on protein level. <b>E:</b> Electrophoretic mobility shift assays (EMSA) showed DNA-binding of c-REL in HeLa Kyoto wt cells (arrow), which was not observable in the <i>c-REL</i> KO clone. <b>F:</b> Immunocytochemistry depicted a nearly complete loss of c-REL-protein in <i>c-REL</i> knockout clone compared to HeLa Kyoto wt cells.</p

Assessment of <i>c-REL</i> overexpression in human cancers and target design of CRISPR/Cas9n-mediated <i>c-REL</i> knockout.

<p><b>A</b>: Database mining revealed more profound overexpression of <b><i>c-</i></b><i>REL</i> in cancers from human ovary, cervix and endometrium compared to oesophagus ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182373#pone.0182373.ref035" target="_blank">35</a>], sancer.sanger.ac.uk; 02-14-2017 16:00; 02-21-2017 15:10). <b>B</b>: Target design showing the proposed <b><i>c-</i></b><i>REL</i> knockout with an expected deletion around 450 bp targeting the intron 1-exon 2-boundary of the <b><i>c-</i></b><i>REL</i> gene. The design was done with the CRISPR/Cas9n Target Online Predictor from the University of Heidelberg [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182373#pone.0182373.ref036" target="_blank">36</a>], crispr.cos.uni-heidelberg.de) and the gene sequence was taken from Ensembl Genome Browser (ensembl.org).</p

<i>c-REL</i> knockout leads to significantly decreased expression levels of NF-κB family member and cell cycle-associated genes.

<p><b>A:</b> qPCR analysis showing significantly decreased mRNA levels of NF-κB family members <i>RELA</i>, <i>NFKB1 (p50)</i>, <i>NFKB2</i> (<i>p52)</i>, <i>IKBKE</i> and <i>TBK1</i> in <i>c-REL</i> knockout cells compared to wildtype cells. <b>B-C:</b> Expression levels of cell cycle-related <i>c-REL</i> target genes <i>A20</i>, <i>BCL2</i>, <i>BCL-XL</i> and <i>TGFB1</i> and <i>c-REL</i> target genes <i>MYC</i> and <i>ICAM-1</i> were significantly decreased in <i>c-REL</i> knockout cells in comparison to HeLa Kyoto wildtype cells. <b>D:</b> Western blot analysis validated the reduced expression levels of RELA and A20 in <i>c-REL</i>^-/- cells in comparison to wt on protein level. WB were performed after TNFα-dependent stimulation of <i>c-REL</i>^-/- and <i>c-REL</i>^+/+ cells. <b>E:</b> Immunocytochemistry revealed a strongly decreased protein amount of ICAM in <i>c-REL</i>^-/- cells in comparison to wt.</p

CRISPR/Cas9-mediated deletion of <i>c-REL</i> results in a decreased proliferation of HeLa Kyoto cell accompanied by strongly reduced amounts of histone H2B.

<p><b>A:</b> Cell number assessed by Orangu Cell Proliferation Assay Kit (Cell Guidance Systems) set against cultivation time showed a strongly increased population doubling time of <i>c-REL</i> knockout cells compared to wt HeLa Kyoto cells. PDT: Population doubling time. <b>B:</b> Flow cytometric DNA content measurements of DAPI-stained <i>c-REL</i> knockout cells showed a decrease in the amount of mitotic cells in <i>c-REL</i> knockout cells compared to wildtype. <b>C:</b> Flow cytometric analysis of Annexin V-stained <i>c-REL</i>^-/- and wt HeLa Kyoto cells revealed only slightly increased amounts of apoptotic cells upon <i>c</i>-REL deletion in comparison to wt. <b>D:</b> Flow cytometric analysis of histone H2B-mCherry showed a strongly decreased amount of the H2B protein in 41.48% of <i>c-REL</i>^-/- HeLa Kyoto cells, which was observable in only 8.67% of HeLa Kyoto wt cells.</p

CRISPR/Cas9-mediated knockout of <i>c-REL</i> in HeLa cells results in profound defects of the cell cycle

<div><p>Cervical cancer is the fourth common cancer in women resulting worldwide in 266,000 deaths per year. Belonging to the carcinomas, new insights into cervical cancer biology may also have great implications for finding new treatment strategies for other kinds of epithelial cancers. Although the transcription factor NF-κB is known as a key player in tumor formation, the relevance of its particular subunits is still underestimated. Here, we applied CRISPR/Cas9n-mediated genome editing to successfully knockout the NF-κB subunit <i>c-REL</i> in HeLa Kyoto cells as a model system for cervical cancers. We successfully generated a homozygous deletion in the <i>c-REL</i> gene, which we validated using sequencing, qPCR, immunocytochemistry, western blot analysis, EMSA and analysis of off-target effects. On the functional level, we observed the deletion of <i>c-REL</i> to result in a significantly decreased cell proliferation in comparison to wildtype (wt) without affecting apoptosis. The impaired proliferative behavior of <i>c-REL</i>^-/- cells was accompanied by a strongly decreased amount of the H2B protein as well as a significant delay in the prometaphase of mitosis compared to <i>c-REL</i>^+/+ HeLa Kyoto cells. <i>c-REL</i>^-/- cells further showed significantly decreased expression levels of <i>c-REL</i> target genes in comparison to wt. In accordance to our proliferation data, we observed the <i>c-REL</i> knockout to result in a significantly increased resistance against the chemotherapeutic agents 5-Fluoro-2’-deoxyuridine (5-FUDR) and cisplatin. In summary, our findings emphasize the importance of c-REL signaling in a cellular model of cervical cancer with direct clinical implications for the development of new treatment strategies.</p></div