AP4 可诱导 JNK1 和 miR-22-3p/FOSL1 前馈环,从而激活 AP-1,促进结直肠癌转移。

IF 20.1 1区 医学 Q1 ONCOLOGY
Jinjiang Chou, Markus Kaller, Matjaz Rokavec, Fangteng Liu, Heiko Hermeking
{"title":"AP4 可诱导 JNK1 和 miR-22-3p/FOSL1 前馈环,从而激活 AP-1,促进结直肠癌转移。","authors":"Jinjiang Chou,&nbsp;Markus Kaller,&nbsp;Matjaz Rokavec,&nbsp;Fangteng Liu,&nbsp;Heiko Hermeking","doi":"10.1002/cac2.12514","DOIUrl":null,"url":null,"abstract":"<p>Dear Editor,</p><p>Colorectal cancer (CRC) is the third most deadly cancer worldwide [<span>1</span>]. The mortality of CRC has remained high due to limited treatment options for metastatic CRC (mCRC) [<span>2</span>]. Epithelial-mesenchymal transition (EMT) is an important contributor to mCRC [<span>2</span>]. The c-MYC proto-oncogene (MYC)-induced transcription factor AP4 (TFAP4/AP4) is a driver of EMT, thereby presumably facilitates mCRC [<span>3, 4</span>]. The mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway has been implicated in the regulation of EMT and mCRC [<span>5</span>].</p><p>Here, we analyzed whether AP4 regulates components of the MAPK/JNK/AP-1 pathway after MYC activation using CRC cells rendered <i>AP4</i>-deficient by a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) approach. The detailed methods are shown in the Supplementary file. First, we grouped MYC-induced changes in mRNA expression observed in the CRC cell line DLD-1 <i>AP4</i> wild-type 1/pRTR-<i>c-MYC</i>-VSV (<i>AP4</i>-WT1 DLD-1/pRTR-<i>c</i>-<i>MYC-</i>VSV) into 6 non-overlapping expression clusters (Supplementary Figure S1A, left), with cluster 1 representing mRNAs down-regulated, and clusters 2-6 representing different patterns of mRNA up-regulated after MYC activation. MAPK signaling pathway components were strongly over-represented in cluster 6 (Supplementary Figure S1A, right). The AP4 targets <i>MIR22 host gene</i> (<i>MIR22HG</i>) and <i>E-</i><i>cadherin 1</i> (<i>CDH1</i>) were down-regulated after MYC activation in <i>AP4</i>-WT1 DLD-1/pRTR-<i>c-MYC</i>-VSV cells (Supplementary Figure S1B). MAPK signaling effectors, including c-<i>Fos proto-oncogene</i> (<i>FOS</i>), c-<i>Jun proto-oncogene</i> (<i>JUN</i>) and <i>c-J</i><i>unB proto-oncogene</i> (<i>JUNB</i>), were over-represented in cluster 6. Additional MAPK signaling pathway components. such as <i>c-Jun N-terminal kinase 1</i> (<i>JNK1</i>), <i>mitogen-activated protein kinase kinase kinase 1</i> (<i>MAP3K1</i>), <i>mitogen-activated protein kinase kinase kinase 13</i> (<i>MAP3K13</i>), <i>mitogen-activated protein kinase kinase 3</i> (<i>MAP2K3</i>), <i>mitogen-activated protein kinase kinase 7</i> (<i>MAP2K7</i>) and <i>FOS</i>-<i>like</i> <i>1</i> (<i>FOSL1</i>) were found in clusters 3-5 (Supplementary Figure S1B). Interestingly, <i>MAP3K13, MAP2K7, JNK1</i> and <i>FOSL1</i> were induced by MYC in an <i>AP4</i>-dependent manner (Supplementary Figure S1C-D).</p><p>Notably, <i>MAP3K13</i>, <i>FOSL1, JNK1</i> and <i>MAP2K7</i> were also up-regulated after activating <i>AP4</i> for 48 or 72 hours (Figure 1A) and showed AP4-binding sites (CAGCTG) and AP4 occupancy (Supplementary Figure S2A). Therefore, these genes presumably represent direct AP4 targets. MAP3K13 and FOSL1 protein and phosphorylated JNK1 were up-regulated after <i>AP4</i> activation, whereas JNK1 protein levels remained unchanged (Figure 1B). Furthermore, AP4 occupancy at the <i>MAP3K13</i> and <i>FOSL1</i> promoters was confirmed by quantitative real-time polymerase chain reaction-chromatin immunoprecipitation (qChIP) analysis (Figure 1C). The nascent mRNA of <i>MAP3K13</i> and <i>FOSL1</i> was decreased in <i>AP4</i>-deficient DLD-1 and SW480 cells (Supplementary Figure S2B). Ectopic AP4 induced nascent <i>MAP3K13</i> and <i>FOSL1</i> mRNA in CRC cells (Supplementary Figure S2C). Therefore, AP4 activates the JNK1 pathway through a MAP3K13/MAP2K7/JNK1 cascade. CRCs with liver metastasis are significantly more often FOSL1-positive [<span>6</span>], and <i>MAP3K13</i> is up-regulated in colon cancer tissues [<span>7</span>]. Thus, AP4-induced <i>FOSL1</i> and <i>MAP3K13</i> are presumably clinically relevant in CRC. <i>FOSL1</i> showed a trend towards a positive correlation with <i>AP4</i> in 471 primary CRC samples of The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) cohort and in an independent patient cohort (GSE13294) (Supplementary Figure S2D). The dimerization of FOSL1 and c-Jun proto-oncogene (JUN) results in the formation of AP-1 [<span>8</span>]. Consistently, AP-1 activity was induced by ectopic AP4 (Figure 1D, Supplementary Figure S2E). In addition, AP-1 activity was significantly lower in <i>AP4</i>-deficient DLD-1 cells (Supplementary Figure S2F). Taken together, these results show that AP4 activates JNK1 and AP-1 by directly inducing <i>MAP3K13</i> and <i>FOSL1</i>.</p><p>Next, we analyzed the relevance of JNK1 for AP4-induced EMT. The JNK1 inhibitor DB07268 partially abrogated the induction of vimentin (<i>VIM</i>) and the repression of <i>CDH1</i> by AP4 in CRC cell lines (Supplementary Figure S3A), indicating that AP4-mediated JNK1 activation contributes to the induction of <i>VIM</i> and the <i>CDH1</i> repression by AP4 [<span>3</span>]. In addition, DB07268 significantly suppressed AP4-induced migration and invasion (Figure 1E, Supplementary Figure S3B-C). Furthermore, DB07268 largely suppressed the AP4-induced proliferation of DLD-1 cells (Supplementary Figure S3D). Therefore, JNK1 activation mediates the induction of EMT, migration, invasion and proliferation by AP4.</p><p>We have previously shown that AP4 represses the tumor-suppressive miRNA miR-22-3p, which contributes to the up-regulation of <i>mediator of DNA damage checkpoint 1</i> (<i>MDC1</i>), a miR-22-3p target, by AP4 [<span>9</span>]. Since we identified a miR-22-3p seed-match sequence (SMS) in the <i>FOSL1</i> 3’-untranslated region (3’-UTR), we investigated whether <i>FOSL1</i> is also regulated by AP4 via repression of miR-22-3p (Figure 1F). Indeed, a <i>FOSL1</i> 3’-UTR reporter was repressed by ectopic miR-22-3p, and mutation of the SMS rendered the reporter refractory to miR-22-3p (Figure 1G). Ectopic miR-22-3p repressed <i>FOSL1</i> (Supplementary Figure S4A) and effectively decreased basal and AP4-induced FOSL1 protein levels (Figure 1H, Supplementary Figure S4B). Next, we generated <i>MIR22</i>-/- (<i>MIR22-</i>knockout [KO]) and <i>FOSL1</i> ΔSMS CRC cell lines (Supplementary Figure S4C). FOSL1 mRNA and protein expression were elevated in <i>MIR22</i>-deficient CRC cells (Figure 1I, Supplementary Figure S4D). <i>MIR22</i>-deficiency increased the activity of the <i>FOSL1</i> 3’-UTR reporter in a miR-22-3p SMS-dependent manner (Supplementary Figure S4E) and enhanced migration and invasion (Supplementary Figure S4F). Furthermore, the intrinsic AP-1 activity was significantly higher in <i>MIR22</i>-deficient CRC cells (Figure 1J, Supplementary Figure S4G). The half-life (t<sub>1/2</sub>) of <i>FOSL1</i> mRNA was prolonged in <i>MIR22</i>-deficient and <i>FOSL1</i> ΔSMS DLD-1 cells (Figure 1K). Ectopic expression of miR-22-3p failed to repress FOSL1 in <i>FOSL1</i> ΔSMS CRC cells (Figure 1L, Supplementary Figure S4H), which showed increased basal FOSL1 expression, and attenuated the up-regulation of FOSL1 by AP4 (Figure 1M). Therefore, the direct targeting of <i>FOSL1</i> by miR-22-3p is essential for maintaining low levels of FOSL1 expression, which allows FOSL1 to be induced by AP4 directly and indirectly via repression of <i>MIR22</i>.</p><p>Next, we determined whether FOSL1 mediates functional effects of AP4 on CRC cells. Ectopic AP4 repressed <i>CDH1</i> and induced <i>VIM</i> in CRC cells, and silencing <i>FOSL1</i> partially compromised these effects (Supplementary Figure S5A). The positive effect of ectopic AP4 on migration and invasion was largely abrogated by <i>FOSL1</i> silencing (Figure 1N, Supplementary Figure S5B-C), indicating that FOSL1 mediates these effects of AP4. Silencing <i>FOSL1</i> significantly repressed AP-1 activity and compromised the effects of AP4 on AP-1 activity (Supplementary Figure S5D). In addition, ectopic AP4 induced the proliferation of DLD-1 cells, which was repressed by concomitant siRNA-mediated silencing of <i>FOSL1</i> (Supplementary Figure S5E). Taken together, these results demonstrate that the FOSL1-mediated activation of AP-1 by AP4 is required for the effects of AP4 on EMT, migration, invasion and proliferation.</p><p>miR-22-3p was shown to be significantly down-regulated in primary CRCs, which displayed lymph node metastasis, and ectopic miR-22-3p repressed lung metastasis formation in a xenograft model [<span>10</span>]. Therefore, deregulation of the miR-22-3p/FOSL1 axis by AP4 may be clinically relevant for mCRC. Notably, AP4 activation in non-metastatic DLD-1 cells allowed these to form lung metastases in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice as determined by non-invasive bioluminescence imaging (Figure 1O-P) and histological analysis (Supplementary Figure S6A, right). Therefore, AP4 activation is sufficient to convert CRC cells from a non-metastatic to a metastatic state. Silencing <i>FOSL1</i> efficiently prevented the AP4-induced formation of lung metastases (Figure 1Q, Supplementary Figure S6A left). Therefore, <i>FOSL1</i> mediates and is required for the formation of AP4-induced metastases. Similar results were obtained after the inhibition of JNK1 using DB07268 (Figure 1R-S, Supplementary Figure S6B-C).</p><p>In conclusion, AP4 activates JNK1 and induces <i>FOSL1</i> directly, as well as indirectly, via repression of miR-22-3p. AP4, miR-22-3p and FOSL1 form a coherent, regulatory feed-forward loop. These regulations converge in the activation of AP-1 (Figure 1T), which ultimately promotes EMT, migration and invasion, thereby enhancing metastasis formation. In the future, these findings may be exploited for the prevention and/or treatment of mCRC.</p><p>Heiko Hermeking conceived, planned and supervised the project; Heiko Hermeking, Jinjiang Chou and Markus Kaller designed experiments; Jinjiang Chou performed experiments and analyzed results; Markus Kaller performed bioinformatics analysis; Matjaz Rokavec performed and analyzed mouse experiments. Fangteng Liu histochemically evaluated lung metastases in mice. Heiko Hermeking, Jinjiang Chou and Markus Kaller wrote the manuscript. All authors read and approved the final manuscript.</p><p>The authors declare that they have no competing interests.</p><p>This work was supported by German Cancer Aid/Deutsche Krebshilfe grants (70114235 and 70112245) to Heiko Hermeking.</p><p>Animal experimentations and analyses were approved by the Government of Upper Bavaria, Germany (55.2-2532.vet_02-18-57).</p><p>Not applicable.</p>","PeriodicalId":9495,"journal":{"name":"Cancer Communications","volume":null,"pages":null},"PeriodicalIF":20.1000,"publicationDate":"2024-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cac2.12514","citationCount":"0","resultStr":"{\"title\":\"AP4 induces JNK1 and a miR-22-3p/FOSL1 feed-forward loop to activate AP-1 and promote colorectal cancer metastasis\",\"authors\":\"Jinjiang Chou,&nbsp;Markus Kaller,&nbsp;Matjaz Rokavec,&nbsp;Fangteng Liu,&nbsp;Heiko Hermeking\",\"doi\":\"10.1002/cac2.12514\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Dear Editor,</p><p>Colorectal cancer (CRC) is the third most deadly cancer worldwide [<span>1</span>]. The mortality of CRC has remained high due to limited treatment options for metastatic CRC (mCRC) [<span>2</span>]. Epithelial-mesenchymal transition (EMT) is an important contributor to mCRC [<span>2</span>]. The c-MYC proto-oncogene (MYC)-induced transcription factor AP4 (TFAP4/AP4) is a driver of EMT, thereby presumably facilitates mCRC [<span>3, 4</span>]. The mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway has been implicated in the regulation of EMT and mCRC [<span>5</span>].</p><p>Here, we analyzed whether AP4 regulates components of the MAPK/JNK/AP-1 pathway after MYC activation using CRC cells rendered <i>AP4</i>-deficient by a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) approach. The detailed methods are shown in the Supplementary file. First, we grouped MYC-induced changes in mRNA expression observed in the CRC cell line DLD-1 <i>AP4</i> wild-type 1/pRTR-<i>c-MYC</i>-VSV (<i>AP4</i>-WT1 DLD-1/pRTR-<i>c</i>-<i>MYC-</i>VSV) into 6 non-overlapping expression clusters (Supplementary Figure S1A, left), with cluster 1 representing mRNAs down-regulated, and clusters 2-6 representing different patterns of mRNA up-regulated after MYC activation. MAPK signaling pathway components were strongly over-represented in cluster 6 (Supplementary Figure S1A, right). The AP4 targets <i>MIR22 host gene</i> (<i>MIR22HG</i>) and <i>E-</i><i>cadherin 1</i> (<i>CDH1</i>) were down-regulated after MYC activation in <i>AP4</i>-WT1 DLD-1/pRTR-<i>c-MYC</i>-VSV cells (Supplementary Figure S1B). MAPK signaling effectors, including c-<i>Fos proto-oncogene</i> (<i>FOS</i>), c-<i>Jun proto-oncogene</i> (<i>JUN</i>) and <i>c-J</i><i>unB proto-oncogene</i> (<i>JUNB</i>), were over-represented in cluster 6. Additional MAPK signaling pathway components. such as <i>c-Jun N-terminal kinase 1</i> (<i>JNK1</i>), <i>mitogen-activated protein kinase kinase kinase 1</i> (<i>MAP3K1</i>), <i>mitogen-activated protein kinase kinase kinase 13</i> (<i>MAP3K13</i>), <i>mitogen-activated protein kinase kinase 3</i> (<i>MAP2K3</i>), <i>mitogen-activated protein kinase kinase 7</i> (<i>MAP2K7</i>) and <i>FOS</i>-<i>like</i> <i>1</i> (<i>FOSL1</i>) were found in clusters 3-5 (Supplementary Figure S1B). Interestingly, <i>MAP3K13, MAP2K7, JNK1</i> and <i>FOSL1</i> were induced by MYC in an <i>AP4</i>-dependent manner (Supplementary Figure S1C-D).</p><p>Notably, <i>MAP3K13</i>, <i>FOSL1, JNK1</i> and <i>MAP2K7</i> were also up-regulated after activating <i>AP4</i> for 48 or 72 hours (Figure 1A) and showed AP4-binding sites (CAGCTG) and AP4 occupancy (Supplementary Figure S2A). Therefore, these genes presumably represent direct AP4 targets. MAP3K13 and FOSL1 protein and phosphorylated JNK1 were up-regulated after <i>AP4</i> activation, whereas JNK1 protein levels remained unchanged (Figure 1B). Furthermore, AP4 occupancy at the <i>MAP3K13</i> and <i>FOSL1</i> promoters was confirmed by quantitative real-time polymerase chain reaction-chromatin immunoprecipitation (qChIP) analysis (Figure 1C). The nascent mRNA of <i>MAP3K13</i> and <i>FOSL1</i> was decreased in <i>AP4</i>-deficient DLD-1 and SW480 cells (Supplementary Figure S2B). Ectopic AP4 induced nascent <i>MAP3K13</i> and <i>FOSL1</i> mRNA in CRC cells (Supplementary Figure S2C). Therefore, AP4 activates the JNK1 pathway through a MAP3K13/MAP2K7/JNK1 cascade. CRCs with liver metastasis are significantly more often FOSL1-positive [<span>6</span>], and <i>MAP3K13</i> is up-regulated in colon cancer tissues [<span>7</span>]. Thus, AP4-induced <i>FOSL1</i> and <i>MAP3K13</i> are presumably clinically relevant in CRC. <i>FOSL1</i> showed a trend towards a positive correlation with <i>AP4</i> in 471 primary CRC samples of The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) cohort and in an independent patient cohort (GSE13294) (Supplementary Figure S2D). The dimerization of FOSL1 and c-Jun proto-oncogene (JUN) results in the formation of AP-1 [<span>8</span>]. Consistently, AP-1 activity was induced by ectopic AP4 (Figure 1D, Supplementary Figure S2E). In addition, AP-1 activity was significantly lower in <i>AP4</i>-deficient DLD-1 cells (Supplementary Figure S2F). Taken together, these results show that AP4 activates JNK1 and AP-1 by directly inducing <i>MAP3K13</i> and <i>FOSL1</i>.</p><p>Next, we analyzed the relevance of JNK1 for AP4-induced EMT. The JNK1 inhibitor DB07268 partially abrogated the induction of vimentin (<i>VIM</i>) and the repression of <i>CDH1</i> by AP4 in CRC cell lines (Supplementary Figure S3A), indicating that AP4-mediated JNK1 activation contributes to the induction of <i>VIM</i> and the <i>CDH1</i> repression by AP4 [<span>3</span>]. In addition, DB07268 significantly suppressed AP4-induced migration and invasion (Figure 1E, Supplementary Figure S3B-C). Furthermore, DB07268 largely suppressed the AP4-induced proliferation of DLD-1 cells (Supplementary Figure S3D). Therefore, JNK1 activation mediates the induction of EMT, migration, invasion and proliferation by AP4.</p><p>We have previously shown that AP4 represses the tumor-suppressive miRNA miR-22-3p, which contributes to the up-regulation of <i>mediator of DNA damage checkpoint 1</i> (<i>MDC1</i>), a miR-22-3p target, by AP4 [<span>9</span>]. Since we identified a miR-22-3p seed-match sequence (SMS) in the <i>FOSL1</i> 3’-untranslated region (3’-UTR), we investigated whether <i>FOSL1</i> is also regulated by AP4 via repression of miR-22-3p (Figure 1F). Indeed, a <i>FOSL1</i> 3’-UTR reporter was repressed by ectopic miR-22-3p, and mutation of the SMS rendered the reporter refractory to miR-22-3p (Figure 1G). Ectopic miR-22-3p repressed <i>FOSL1</i> (Supplementary Figure S4A) and effectively decreased basal and AP4-induced FOSL1 protein levels (Figure 1H, Supplementary Figure S4B). Next, we generated <i>MIR22</i>-/- (<i>MIR22-</i>knockout [KO]) and <i>FOSL1</i> ΔSMS CRC cell lines (Supplementary Figure S4C). FOSL1 mRNA and protein expression were elevated in <i>MIR22</i>-deficient CRC cells (Figure 1I, Supplementary Figure S4D). <i>MIR22</i>-deficiency increased the activity of the <i>FOSL1</i> 3’-UTR reporter in a miR-22-3p SMS-dependent manner (Supplementary Figure S4E) and enhanced migration and invasion (Supplementary Figure S4F). Furthermore, the intrinsic AP-1 activity was significantly higher in <i>MIR22</i>-deficient CRC cells (Figure 1J, Supplementary Figure S4G). The half-life (t<sub>1/2</sub>) of <i>FOSL1</i> mRNA was prolonged in <i>MIR22</i>-deficient and <i>FOSL1</i> ΔSMS DLD-1 cells (Figure 1K). Ectopic expression of miR-22-3p failed to repress FOSL1 in <i>FOSL1</i> ΔSMS CRC cells (Figure 1L, Supplementary Figure S4H), which showed increased basal FOSL1 expression, and attenuated the up-regulation of FOSL1 by AP4 (Figure 1M). Therefore, the direct targeting of <i>FOSL1</i> by miR-22-3p is essential for maintaining low levels of FOSL1 expression, which allows FOSL1 to be induced by AP4 directly and indirectly via repression of <i>MIR22</i>.</p><p>Next, we determined whether FOSL1 mediates functional effects of AP4 on CRC cells. Ectopic AP4 repressed <i>CDH1</i> and induced <i>VIM</i> in CRC cells, and silencing <i>FOSL1</i> partially compromised these effects (Supplementary Figure S5A). The positive effect of ectopic AP4 on migration and invasion was largely abrogated by <i>FOSL1</i> silencing (Figure 1N, Supplementary Figure S5B-C), indicating that FOSL1 mediates these effects of AP4. Silencing <i>FOSL1</i> significantly repressed AP-1 activity and compromised the effects of AP4 on AP-1 activity (Supplementary Figure S5D). In addition, ectopic AP4 induced the proliferation of DLD-1 cells, which was repressed by concomitant siRNA-mediated silencing of <i>FOSL1</i> (Supplementary Figure S5E). Taken together, these results demonstrate that the FOSL1-mediated activation of AP-1 by AP4 is required for the effects of AP4 on EMT, migration, invasion and proliferation.</p><p>miR-22-3p was shown to be significantly down-regulated in primary CRCs, which displayed lymph node metastasis, and ectopic miR-22-3p repressed lung metastasis formation in a xenograft model [<span>10</span>]. Therefore, deregulation of the miR-22-3p/FOSL1 axis by AP4 may be clinically relevant for mCRC. Notably, AP4 activation in non-metastatic DLD-1 cells allowed these to form lung metastases in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice as determined by non-invasive bioluminescence imaging (Figure 1O-P) and histological analysis (Supplementary Figure S6A, right). Therefore, AP4 activation is sufficient to convert CRC cells from a non-metastatic to a metastatic state. Silencing <i>FOSL1</i> efficiently prevented the AP4-induced formation of lung metastases (Figure 1Q, Supplementary Figure S6A left). Therefore, <i>FOSL1</i> mediates and is required for the formation of AP4-induced metastases. Similar results were obtained after the inhibition of JNK1 using DB07268 (Figure 1R-S, Supplementary Figure S6B-C).</p><p>In conclusion, AP4 activates JNK1 and induces <i>FOSL1</i> directly, as well as indirectly, via repression of miR-22-3p. AP4, miR-22-3p and FOSL1 form a coherent, regulatory feed-forward loop. These regulations converge in the activation of AP-1 (Figure 1T), which ultimately promotes EMT, migration and invasion, thereby enhancing metastasis formation. In the future, these findings may be exploited for the prevention and/or treatment of mCRC.</p><p>Heiko Hermeking conceived, planned and supervised the project; Heiko Hermeking, Jinjiang Chou and Markus Kaller designed experiments; Jinjiang Chou performed experiments and analyzed results; Markus Kaller performed bioinformatics analysis; Matjaz Rokavec performed and analyzed mouse experiments. Fangteng Liu histochemically evaluated lung metastases in mice. Heiko Hermeking, Jinjiang Chou and Markus Kaller wrote the manuscript. All authors read and approved the final manuscript.</p><p>The authors declare that they have no competing interests.</p><p>This work was supported by German Cancer Aid/Deutsche Krebshilfe grants (70114235 and 70112245) to Heiko Hermeking.</p><p>Animal experimentations and analyses were approved by the Government of Upper Bavaria, Germany (55.2-2532.vet_02-18-57).</p><p>Not applicable.</p>\",\"PeriodicalId\":9495,\"journal\":{\"name\":\"Cancer Communications\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":20.1000,\"publicationDate\":\"2024-01-15\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cac2.12514\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cancer Communications\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cac2.12514\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"ONCOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cancer Communications","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cac2.12514","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ONCOLOGY","Score":null,"Total":0}
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摘要

由于我们在 FOSL1 3'- 非翻译区(3'-UTR)发现了 miR-22-3p 种子匹配序列(SMS),因此我们研究了 FOSL1 是否也通过抑制 miR-22-3p 受 AP4 的调控(图 1F)。事实上,FOSL1 3'-UTR 报告受异位 miR-22-3p 的抑制,而 SMS 的突变使报告对 miR-22-3p 难以承受(图 1G)。异位 miR-22-3p 抑制了 FOSL1(补充图 S4A),并有效降低了基础和 AP4 诱导的 FOSL1 蛋白水平(图 1H,补充图 S4B)。接下来,我们生成了 MIR22-/- (MIR22-基因剔除 [KO])和 FOSL1 ΔSMS CRC 细胞系(补充图 S4C)。在 MIR22 缺失的 CRC 细胞中,FOSL1 mRNA 和蛋白表达均升高(图 1I,补图 S4D)。MIR22 缺失以 miR-22-3p SMS 依赖性方式增加了 FOSL1 3'-UTR 报告的活性(补充图 S4E),并增强了迁移和侵袭(补充图 S4F)。此外,在 MIR22 缺失的 CRC 细胞中,固有 AP-1 活性显著升高(图 1J,补图 S4G)。MIR22 缺陷和 FOSL1 ΔSMS DLD-1 细胞中 FOSL1 mRNA 的半衰期(t1/2)延长(图 1K)。在 FOSL1 ΔSMS CRC 细胞中异位表达 miR-22-3p 未能抑制 FOSL1(图 1L,补充图 S4H),这表明 FOSL1 的基础表达增加,并减弱了 AP4 对 FOSL1 的上调(图 1M)。因此,miR-22-3p对FOSL1的直接靶向作用对于维持低水平的FOSL1表达至关重要,这使得FOSL1可以被AP4直接诱导,并通过抑制MIR22间接诱导。异位 AP4 可抑制 CDH1 并诱导 CRC 细胞中的 VIM,而沉默 FOSL1 则会部分削弱这些效应(补充图 S5A)。异位AP4对迁移和侵袭的积极作用在很大程度上被FOSL1沉默所削弱(图1N,补充图S5B-C),这表明FOSL1介导了AP4的这些作用。沉默 FOSL1 能显著抑制 AP-1 活性,并削弱 AP4 对 AP-1 活性的影响(补充图 S5D)。此外,异位 AP4 会诱导 DLD-1 细胞增殖,而同时 siRNA 介导的 FOSL1 沉默会抑制这种增殖(补充图 S5E)。综上所述,这些结果表明,AP4对EMT、迁移、侵袭和增殖的影响需要FOSL1介导的AP-1激活。因此,AP4对miR-22-3p/FOSL1轴的调控可能与mCRC的临床相关。值得注意的是,通过非侵入性生物发光成像(图 1O-P)和组织学分析(补充图 S6A,右图)确定,非转移性 DLD-1 细胞中的 AP4 激活可使这些细胞在非肥胖糖尿病/严重联合免疫缺陷(NOD/SCID)小鼠体内形成肺转移。因此,AP4 的激活足以使 CRC 细胞从非转移状态转变为转移状态。沉默 FOSL1 能有效阻止 AP4 诱导的肺转移的形成(图 1Q,补充图 S6A 左)。因此,FOSL1介导了AP4诱导的转移的形成,并且是形成转移所必需的。使用 DB07268 抑制 JNK1 后也得到了类似的结果(图 1R-S,补充图 S6B-C)。AP4、miR-22-3p 和 FOSL1 形成了一个连贯的前馈调节环。这些调控汇聚在 AP-1 的激活上(图 1T),最终促进了 EMT、迁移和侵袭,从而加强了转移的形成。Heiko Hermeking构思、规划并指导了该项目;Heiko Hermeking、周锦江和Markus Kaller设计了实验;周锦江进行了实验并分析了结果;Markus Kaller进行了生物信息学分析;Matjaz Rokavec进行了小鼠实验并分析了结果。刘方腾对小鼠肺转移进行了组织化学评估。Heiko Hermeking、周锦江和Markus Kaller撰写了手稿。动物实验和分析获得了德国上巴伐利亚州政府的批准(55.2-2532.vet_02-18-57)。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

AP4 induces JNK1 and a miR-22-3p/FOSL1 feed-forward loop to activate AP-1 and promote colorectal cancer metastasis

AP4 induces JNK1 and a miR-22-3p/FOSL1 feed-forward loop to activate AP-1 and promote colorectal cancer metastasis

Dear Editor,

Colorectal cancer (CRC) is the third most deadly cancer worldwide [1]. The mortality of CRC has remained high due to limited treatment options for metastatic CRC (mCRC) [2]. Epithelial-mesenchymal transition (EMT) is an important contributor to mCRC [2]. The c-MYC proto-oncogene (MYC)-induced transcription factor AP4 (TFAP4/AP4) is a driver of EMT, thereby presumably facilitates mCRC [3, 4]. The mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway has been implicated in the regulation of EMT and mCRC [5].

Here, we analyzed whether AP4 regulates components of the MAPK/JNK/AP-1 pathway after MYC activation using CRC cells rendered AP4-deficient by a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) approach. The detailed methods are shown in the Supplementary file. First, we grouped MYC-induced changes in mRNA expression observed in the CRC cell line DLD-1 AP4 wild-type 1/pRTR-c-MYC-VSV (AP4-WT1 DLD-1/pRTR-c-MYC-VSV) into 6 non-overlapping expression clusters (Supplementary Figure S1A, left), with cluster 1 representing mRNAs down-regulated, and clusters 2-6 representing different patterns of mRNA up-regulated after MYC activation. MAPK signaling pathway components were strongly over-represented in cluster 6 (Supplementary Figure S1A, right). The AP4 targets MIR22 host gene (MIR22HG) and E-cadherin 1 (CDH1) were down-regulated after MYC activation in AP4-WT1 DLD-1/pRTR-c-MYC-VSV cells (Supplementary Figure S1B). MAPK signaling effectors, including c-Fos proto-oncogene (FOS), c-Jun proto-oncogene (JUN) and c-JunB proto-oncogene (JUNB), were over-represented in cluster 6. Additional MAPK signaling pathway components. such as c-Jun N-terminal kinase 1 (JNK1), mitogen-activated protein kinase kinase kinase 1 (MAP3K1), mitogen-activated protein kinase kinase kinase 13 (MAP3K13), mitogen-activated protein kinase kinase 3 (MAP2K3), mitogen-activated protein kinase kinase 7 (MAP2K7) and FOS-like 1 (FOSL1) were found in clusters 3-5 (Supplementary Figure S1B). Interestingly, MAP3K13, MAP2K7, JNK1 and FOSL1 were induced by MYC in an AP4-dependent manner (Supplementary Figure S1C-D).

Notably, MAP3K13, FOSL1, JNK1 and MAP2K7 were also up-regulated after activating AP4 for 48 or 72 hours (Figure 1A) and showed AP4-binding sites (CAGCTG) and AP4 occupancy (Supplementary Figure S2A). Therefore, these genes presumably represent direct AP4 targets. MAP3K13 and FOSL1 protein and phosphorylated JNK1 were up-regulated after AP4 activation, whereas JNK1 protein levels remained unchanged (Figure 1B). Furthermore, AP4 occupancy at the MAP3K13 and FOSL1 promoters was confirmed by quantitative real-time polymerase chain reaction-chromatin immunoprecipitation (qChIP) analysis (Figure 1C). The nascent mRNA of MAP3K13 and FOSL1 was decreased in AP4-deficient DLD-1 and SW480 cells (Supplementary Figure S2B). Ectopic AP4 induced nascent MAP3K13 and FOSL1 mRNA in CRC cells (Supplementary Figure S2C). Therefore, AP4 activates the JNK1 pathway through a MAP3K13/MAP2K7/JNK1 cascade. CRCs with liver metastasis are significantly more often FOSL1-positive [6], and MAP3K13 is up-regulated in colon cancer tissues [7]. Thus, AP4-induced FOSL1 and MAP3K13 are presumably clinically relevant in CRC. FOSL1 showed a trend towards a positive correlation with AP4 in 471 primary CRC samples of The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) cohort and in an independent patient cohort (GSE13294) (Supplementary Figure S2D). The dimerization of FOSL1 and c-Jun proto-oncogene (JUN) results in the formation of AP-1 [8]. Consistently, AP-1 activity was induced by ectopic AP4 (Figure 1D, Supplementary Figure S2E). In addition, AP-1 activity was significantly lower in AP4-deficient DLD-1 cells (Supplementary Figure S2F). Taken together, these results show that AP4 activates JNK1 and AP-1 by directly inducing MAP3K13 and FOSL1.

Next, we analyzed the relevance of JNK1 for AP4-induced EMT. The JNK1 inhibitor DB07268 partially abrogated the induction of vimentin (VIM) and the repression of CDH1 by AP4 in CRC cell lines (Supplementary Figure S3A), indicating that AP4-mediated JNK1 activation contributes to the induction of VIM and the CDH1 repression by AP4 [3]. In addition, DB07268 significantly suppressed AP4-induced migration and invasion (Figure 1E, Supplementary Figure S3B-C). Furthermore, DB07268 largely suppressed the AP4-induced proliferation of DLD-1 cells (Supplementary Figure S3D). Therefore, JNK1 activation mediates the induction of EMT, migration, invasion and proliferation by AP4.

We have previously shown that AP4 represses the tumor-suppressive miRNA miR-22-3p, which contributes to the up-regulation of mediator of DNA damage checkpoint 1 (MDC1), a miR-22-3p target, by AP4 [9]. Since we identified a miR-22-3p seed-match sequence (SMS) in the FOSL1 3’-untranslated region (3’-UTR), we investigated whether FOSL1 is also regulated by AP4 via repression of miR-22-3p (Figure 1F). Indeed, a FOSL1 3’-UTR reporter was repressed by ectopic miR-22-3p, and mutation of the SMS rendered the reporter refractory to miR-22-3p (Figure 1G). Ectopic miR-22-3p repressed FOSL1 (Supplementary Figure S4A) and effectively decreased basal and AP4-induced FOSL1 protein levels (Figure 1H, Supplementary Figure S4B). Next, we generated MIR22-/- (MIR22-knockout [KO]) and FOSL1 ΔSMS CRC cell lines (Supplementary Figure S4C). FOSL1 mRNA and protein expression were elevated in MIR22-deficient CRC cells (Figure 1I, Supplementary Figure S4D). MIR22-deficiency increased the activity of the FOSL1 3’-UTR reporter in a miR-22-3p SMS-dependent manner (Supplementary Figure S4E) and enhanced migration and invasion (Supplementary Figure S4F). Furthermore, the intrinsic AP-1 activity was significantly higher in MIR22-deficient CRC cells (Figure 1J, Supplementary Figure S4G). The half-life (t1/2) of FOSL1 mRNA was prolonged in MIR22-deficient and FOSL1 ΔSMS DLD-1 cells (Figure 1K). Ectopic expression of miR-22-3p failed to repress FOSL1 in FOSL1 ΔSMS CRC cells (Figure 1L, Supplementary Figure S4H), which showed increased basal FOSL1 expression, and attenuated the up-regulation of FOSL1 by AP4 (Figure 1M). Therefore, the direct targeting of FOSL1 by miR-22-3p is essential for maintaining low levels of FOSL1 expression, which allows FOSL1 to be induced by AP4 directly and indirectly via repression of MIR22.

Next, we determined whether FOSL1 mediates functional effects of AP4 on CRC cells. Ectopic AP4 repressed CDH1 and induced VIM in CRC cells, and silencing FOSL1 partially compromised these effects (Supplementary Figure S5A). The positive effect of ectopic AP4 on migration and invasion was largely abrogated by FOSL1 silencing (Figure 1N, Supplementary Figure S5B-C), indicating that FOSL1 mediates these effects of AP4. Silencing FOSL1 significantly repressed AP-1 activity and compromised the effects of AP4 on AP-1 activity (Supplementary Figure S5D). In addition, ectopic AP4 induced the proliferation of DLD-1 cells, which was repressed by concomitant siRNA-mediated silencing of FOSL1 (Supplementary Figure S5E). Taken together, these results demonstrate that the FOSL1-mediated activation of AP-1 by AP4 is required for the effects of AP4 on EMT, migration, invasion and proliferation.

miR-22-3p was shown to be significantly down-regulated in primary CRCs, which displayed lymph node metastasis, and ectopic miR-22-3p repressed lung metastasis formation in a xenograft model [10]. Therefore, deregulation of the miR-22-3p/FOSL1 axis by AP4 may be clinically relevant for mCRC. Notably, AP4 activation in non-metastatic DLD-1 cells allowed these to form lung metastases in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice as determined by non-invasive bioluminescence imaging (Figure 1O-P) and histological analysis (Supplementary Figure S6A, right). Therefore, AP4 activation is sufficient to convert CRC cells from a non-metastatic to a metastatic state. Silencing FOSL1 efficiently prevented the AP4-induced formation of lung metastases (Figure 1Q, Supplementary Figure S6A left). Therefore, FOSL1 mediates and is required for the formation of AP4-induced metastases. Similar results were obtained after the inhibition of JNK1 using DB07268 (Figure 1R-S, Supplementary Figure S6B-C).

In conclusion, AP4 activates JNK1 and induces FOSL1 directly, as well as indirectly, via repression of miR-22-3p. AP4, miR-22-3p and FOSL1 form a coherent, regulatory feed-forward loop. These regulations converge in the activation of AP-1 (Figure 1T), which ultimately promotes EMT, migration and invasion, thereby enhancing metastasis formation. In the future, these findings may be exploited for the prevention and/or treatment of mCRC.

Heiko Hermeking conceived, planned and supervised the project; Heiko Hermeking, Jinjiang Chou and Markus Kaller designed experiments; Jinjiang Chou performed experiments and analyzed results; Markus Kaller performed bioinformatics analysis; Matjaz Rokavec performed and analyzed mouse experiments. Fangteng Liu histochemically evaluated lung metastases in mice. Heiko Hermeking, Jinjiang Chou and Markus Kaller wrote the manuscript. All authors read and approved the final manuscript.

The authors declare that they have no competing interests.

This work was supported by German Cancer Aid/Deutsche Krebshilfe grants (70114235 and 70112245) to Heiko Hermeking.

Animal experimentations and analyses were approved by the Government of Upper Bavaria, Germany (55.2-2532.vet_02-18-57).

Not applicable.

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来源期刊
Cancer Communications
Cancer Communications Biochemistry, Genetics and Molecular Biology-Cancer Research
CiteScore
25.50
自引率
4.30%
发文量
153
审稿时长
4 weeks
期刊介绍: Cancer Communications is an open access, peer-reviewed online journal that encompasses basic, clinical, and translational cancer research. The journal welcomes submissions concerning clinical trials, epidemiology, molecular and cellular biology, and genetics.
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