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Research
Zuguo Li
ShenZhen Hospital of Southern Medical University
Corresponding Author
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This work is licensed under a CC BY 4.0 License. Read Full License
Background
CIP4 (Cdc42-interacting protein 4), a member of the F-BAR family which plays an important role in regulating cell membrane and actin, has been reported to interact with Cdc42 and closely associated with tumor invadopodia formation. However, the specific mechanism of the interaction between CIP4 and Cdc42 as well as the downstream signaling pathway in response in colorectal cancer (CRC) remains unknown, which is worth exploring for its impact on tumor infiltration and metastasis.
Methods
Immunohistochemistry and western blot analyses were performed to detect the expression of CIP4 and Cdc42. Their relationship with CRC clinicopathological characteristics was further analyzed. Wound-healing, transwell migration and invasion assays tested the effect of CIP4 on cells migration and invasion ability in vitro, and the orthotopic xenograft colorectal cancer mouse mode evaluated the tumor metastasis in vivo. The invadopodia formation and function were assessed by immunofluorescence, scanning electron microscopy (SEM) and matrix degradation assay. The interaction between CIP4 and Cdc42 was confirmed by co-immunoprecipitation (co-IP) and GST-Pull down assays. Immunofluorescence was used to observed the colocalization of CIP4, GTP-Cdc42 and invadopodia. The related downstream signaling pathway was investigated by western blot and immunofluorescence.
Results
CIP4 expression was significantly higher in human colorectal cancer tissues and correlated with the CRC infiltrating depth and metastasis as well as the lower survival rate in patients. In cultured CRC cells, knockdown of CIP4 inhibited cell migration and invasion ability in vitro and the tumor metastasis in vivo, while overexpression of CIP4 confirmed the opposite situation by promoting invadopodia formation and matrix degradation ability. In addition, we identified GTP-Cdc42 as a directly interactive protein of CIP4, which was upregulated and recruited by CIP4 to participate in this process. Furthermore, activated NF-κB signaling pathway was found in CIP4 overexpression CRC cells contributing to invadopodia formation while inhibition of either CIP4 or Cdc42 led to suppression of NF-κB pathway resulted in decrease quantity of invadopodia.
Conclusion
Our findings suggested that CIP4 targets to recruit GTP-Cdc42 and directly combines with it to accelerate invadopodia formation and function by activating NF-κB signaling pathway, thus promoting CRC infiltration and metastasis.
Keywords
CIP4, Cdc42, NF-κB, invadopodia, colorectal cancer, invasion and metastasis
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1.tif
Figure S1. CIP4 plays an important role in invadopodia formation in CRC cells. (A) Quantification of cells with invadopodia was analyzed by immunofluorescence. White arrowhead indicates the invadopodia. Error bars represent the mean ± S.D (n=200 cells). ***P<0.001. Magnification: 60x. (B) The morphology of invadopdia in CRC cells shown by confocal laser scanning. Magnification: 180x.
- Additionalfile2.tif
Figure S2. CIP4 directly interacts with activated Cdc42. (A) Co-localization of CIP4 (green) and Cdc42 (red) in CIP4 over-expressing HCT116 and DLD1 cells was observed by immunofluorescence. Magnification: 180x. The Person's correlation and overlap coefficient were shown in bar graph format. Error bars represent the mean±S.D (n=5). (B) Coomassie brilliant blue staining showed the optimal concentration and time of CIP4-GST and L61Cdc42-6HIS fusion protein expression induced by IPTG. (C) GST-Pull down experiment of CIP4 protein with mutant of activated Cdc42. Coomassie brilliant blue staining showed the protein bands.
- Additionalfile3.tif
Figure S3. The optimum conditions of inhibitors and activator to treat cells. (A-B) Western blot analyzed the optimal time of NF-κB inhibitor QNZ and Cdc42 inhibitor ML141 acting on CIP4-overexpression HCT116 and DLD1 cells. The indicated concentrations of QNZ and ML141 are recommended in the specifications. DMSO is the solvent of QNZ and ML141 and treated cells as control. (C) Western blot analyzed the optimal time of NF-κB activator LPS acting on CIP4-knockdown Lovo and HT29 cells. The indicated concentration is recommended in the specification.
- Additionalfile4.tif
Figure S4. The NF-κB signaling pathway is involved in accelerating invadopodia formation regulated by CIP4 through GTP-Cdc42. (A-B) Western blot analyzed the expression of CIP4, Cdc42, GTP-Cdc42, p65, p-p65 and p65 in nucleus in HT29 (the CIP4-knockdown cells were treated with LPS, 10 μg/ml for 0.5h) and DLD1 (the CIP4-overexpression cells were treated with ML141, 20μM for 24h, or QNZ, 10μM for 16h) cells as indicated. Grayscale values were normalized to GAPDH and histone H3 in the nucleus. (C-D) Quantification of cells with invadopodia was analyzed by immunofluorescence. HT29 CIP4-knockdown cells were treated with LPS, 10 μg/ml for 0.5h, DLD1 CIP4-overexpression cells were treated with ML141, 20μM for 24h, or QNZ, 10μM for 16h. White arrowhead indicates the invadopodia. Error bars represent the mean ± S.D (HT29: P=0.0012, DLD1: P=0.0021, n=200 cells). Magnification: 60x.
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Zuguo Li
ShenZhen Hospital of Southern Medical University
Corresponding Author
Badges
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Complete author information
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CURRENT STATUS: Posted
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License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
CIP4 (Cdc42-interacting protein 4), a member of the F-BAR family which plays an important role in regulating cell membrane and actin, has been reported to interact with Cdc42 and closely associated with tumor invadopodia formation. However, the specific mechanism of the interaction between CIP4 and Cdc42 as well as the downstream signaling pathway in response in colorectal cancer (CRC) remains unknown, which is worth exploring for its impact on tumor infiltration and metastasis.
Methods
Immunohistochemistry and western blot analyses were performed to detect the expression of CIP4 and Cdc42. Their relationship with CRC clinicopathological characteristics was further analyzed. Wound-healing, transwell migration and invasion assays tested the effect of CIP4 on cells migration and invasion ability in vitro, and the orthotopic xenograft colorectal cancer mouse mode evaluated the tumor metastasis in vivo. The invadopodia formation and function were assessed by immunofluorescence, scanning electron microscopy (SEM) and matrix degradation assay. The interaction between CIP4 and Cdc42 was confirmed by co-immunoprecipitation (co-IP) and GST-Pull down assays. Immunofluorescence was used to observed the colocalization of CIP4, GTP-Cdc42 and invadopodia. The related downstream signaling pathway was investigated by western blot and immunofluorescence.
Results
CIP4 expression was significantly higher in human colorectal cancer tissues and correlated with the CRC infiltrating depth and metastasis as well as the lower survival rate in patients. In cultured CRC cells, knockdown of CIP4 inhibited cell migration and invasion ability in vitro and the tumor metastasis in vivo, while overexpression of CIP4 confirmed the opposite situation by promoting invadopodia formation and matrix degradation ability. In addition, we identified GTP-Cdc42 as a directly interactive protein of CIP4, which was upregulated and recruited by CIP4 to participate in this process. Furthermore, activated NF-κB signaling pathway was found in CIP4 overexpression CRC cells contributing to invadopodia formation while inhibition of either CIP4 or Cdc42 led to suppression of NF-κB pathway resulted in decrease quantity of invadopodia.
Conclusion
Our findings suggested that CIP4 targets to recruit GTP-Cdc42 and directly combines with it to accelerate invadopodia formation and function by activating NF-κB signaling pathway, thus promoting CRC infiltration and metastasis.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1.tif
Figure S1. CIP4 plays an important role in invadopodia formation in CRC cells. (A) Quantification of cells with invadopodia was analyzed by immunofluorescence. White arrowhead indicates the invadopodia. Error bars represent the mean ± S.D (n=200 cells). ***P<0.001. Magnification: 60x. (B) The morphology of invadopdia in CRC cells shown by confocal laser scanning. Magnification: 180x.
- Additionalfile2.tif
Figure S2. CIP4 directly interacts with activated Cdc42. (A) Co-localization of CIP4 (green) and Cdc42 (red) in CIP4 over-expressing HCT116 and DLD1 cells was observed by immunofluorescence. Magnification: 180x. The Person's correlation and overlap coefficient were shown in bar graph format. Error bars represent the mean±S.D (n=5). (B) Coomassie brilliant blue staining showed the optimal concentration and time of CIP4-GST and L61Cdc42-6HIS fusion protein expression induced by IPTG. (C) GST-Pull down experiment of CIP4 protein with mutant of activated Cdc42. Coomassie brilliant blue staining showed the protein bands.
- Additionalfile3.tif
Figure S3. The optimum conditions of inhibitors and activator to treat cells. (A-B) Western blot analyzed the optimal time of NF-κB inhibitor QNZ and Cdc42 inhibitor ML141 acting on CIP4-overexpression HCT116 and DLD1 cells. The indicated concentrations of QNZ and ML141 are recommended in the specifications. DMSO is the solvent of QNZ and ML141 and treated cells as control. (C) Western blot analyzed the optimal time of NF-κB activator LPS acting on CIP4-knockdown Lovo and HT29 cells. The indicated concentration is recommended in the specification.
- Additionalfile4.tif
Figure S4. The NF-κB signaling pathway is involved in accelerating invadopodia formation regulated by CIP4 through GTP-Cdc42. (A-B) Western blot analyzed the expression of CIP4, Cdc42, GTP-Cdc42, p65, p-p65 and p65 in nucleus in HT29 (the CIP4-knockdown cells were treated with LPS, 10 μg/ml for 0.5h) and DLD1 (the CIP4-overexpression cells were treated with ML141, 20μM for 24h, or QNZ, 10μM for 16h) cells as indicated. Grayscale values were normalized to GAPDH and histone H3 in the nucleus. (C-D) Quantification of cells with invadopodia was analyzed by immunofluorescence. HT29 CIP4-knockdown cells were treated with LPS, 10 μg/ml for 0.5h, DLD1 CIP4-overexpression cells were treated with ML141, 20μM for 24h, or QNZ, 10μM for 16h. White arrowhead indicates the invadopodia. Error bars represent the mean ± S.D (HT29: P=0.0012, DLD1: P=0.0021, n=200 cells). Magnification: 60x.
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