Limits...
Role of MUC4-NIDO domain in the MUC4-mediated metastasis of pancreatic cancer cells.

Senapati S, Gnanapragassam VS, Moniaux N, Momi N, Batra SK - Oncogene (2011)

Bottom Line: The in vitro studies demonstrated an enhanced invasiveness of MiaPaCa cells expressing MUC4 (MiaPaCa-MUC4) compared with vector-transfected cells (MiaPaCa-Vec; P=0.003) or cells expressing MUC4 without the NIDO domain (MiaPaCa-MUC4-NIDO(Δ); P=0.03).However, the absence of NIDO-domain has no significant role on cell growth and motility (P=0.93).Additionally, a reduced binding (P=0.0004) of MiaPaCa-MUC4-NIDO(Δ) cells to the fibulin-2 coated plates compared with MiaPaCa-MUC4 cells indicated a possible interaction between the MUC4-NIDO domain and fibulin-2, a nidogen-interacting protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.

ABSTRACT
MUC4 is a large transmembrane type I glycoprotein that is overexpressed in pancreatic cancer (PC) and has been shown to be associated with its progression and metastasis. However, the exact cellular and molecular mechanism(s) through which MUC4 promotes metastasis of PC cells has been sparsely studied. Here we showed that the nidogen-like (NIDO) domain of MUC4, which is similar to the G1-domain present in the nidogen or entactin (an extracellular matrix protein), contributes to the protein-protein interaction property of MUC4. By this interaction, MUC4 promotes breaching of basement membrane (BM) integrity, and spreading of cancer cells. These observations are corroborated with the data from our study using an engineered MUC4 protein without the NIDO domain, which was ectopically expressed in the MiaPaCa PC cells, lacking endogenous MUC4 and nidogen protein. The in vitro studies demonstrated an enhanced invasiveness of MiaPaCa cells expressing MUC4 (MiaPaCa-MUC4) compared with vector-transfected cells (MiaPaCa-Vec; P=0.003) or cells expressing MUC4 without the NIDO domain (MiaPaCa-MUC4-NIDO(Δ); P=0.03). However, the absence of NIDO-domain has no significant role on cell growth and motility (P=0.93). In the in vivo studies, all the mice orthotopically implanted with MiPaCa-MUC4 cells developed metastasis to the liver as compared with MiaPaCa-Vec or the MiaPaCa-MUC4-NIDO(Δ) group, hence, supporting our in vitro observations. Additionally, a reduced binding (P=0.0004) of MiaPaCa-MUC4-NIDO(Δ) cells to the fibulin-2 coated plates compared with MiaPaCa-MUC4 cells indicated a possible interaction between the MUC4-NIDO domain and fibulin-2, a nidogen-interacting protein. Furthermore, in PC tissue samples, MUC4 colocalized with the fibulin-2 present in the BM. Altogether, our findings demonstrate that the MUC4-NIDO domain significantly contributes to the MUC4-mediated metastasis of PC cells. This may be partly due to the interaction between the MUC4-NIDO domain and fibulin-2.

Show MeSH

Related in: MedlinePlus

MUC4 NIDO domain interactions with Fibulin-2 in vitro, and colocalisation in pancreatic tissue(A) Immunohistofluorescence analysis was carried out on formalin-fixed and paraffin-embedded pancreatic tumor tissue sections after rehydration and antigen-retrieval. Digital merging of the immunostained tissue section clearly showed the colocalization of MUC4 (green) and fibulin-2 (red) in an adenocarcinoma lesion. Absence of MUC4 and fibulin-2 staining in the endocrine glands of the same tissue section acted as a negative control. (B) Binding Assay Cells (25 × 103) were seeded in 96-well plate coated with fibulin-2. Wells coated with bovine serum albumin and poly-L-lysine served as negative and positive controls, respectively. A total of 100 μl of the cell suspension was seeded in triplicates onto the fibulin-2-coated 96 well plates and incubated for 1 h at 37°C. Next the adhered cells were labeled with DIO dye for 1 h at 37°C. The fluorescence of the samples was measured by using the fluorescence plate reader at an excitation wavelength of 484 nm and an emission wavelength of 501 nm. The fluorescence intensity obtained in the negative control (BSA) was subtracted from the values from fibulin-2 coated plates. A relative fluorescence (RFU) was calculated for all treatments with respect to the intensity value obtained with the positive control (poly-L-lysine-coated plates). The significance of each binding assay was evaluated using the t test assuming unequal variances. P-values lower than 0.05 were considered statistically significant.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3298579&req=5

Figure 5: MUC4 NIDO domain interactions with Fibulin-2 in vitro, and colocalisation in pancreatic tissue(A) Immunohistofluorescence analysis was carried out on formalin-fixed and paraffin-embedded pancreatic tumor tissue sections after rehydration and antigen-retrieval. Digital merging of the immunostained tissue section clearly showed the colocalization of MUC4 (green) and fibulin-2 (red) in an adenocarcinoma lesion. Absence of MUC4 and fibulin-2 staining in the endocrine glands of the same tissue section acted as a negative control. (B) Binding Assay Cells (25 × 103) were seeded in 96-well plate coated with fibulin-2. Wells coated with bovine serum albumin and poly-L-lysine served as negative and positive controls, respectively. A total of 100 μl of the cell suspension was seeded in triplicates onto the fibulin-2-coated 96 well plates and incubated for 1 h at 37°C. Next the adhered cells were labeled with DIO dye for 1 h at 37°C. The fluorescence of the samples was measured by using the fluorescence plate reader at an excitation wavelength of 484 nm and an emission wavelength of 501 nm. The fluorescence intensity obtained in the negative control (BSA) was subtracted from the values from fibulin-2 coated plates. A relative fluorescence (RFU) was calculated for all treatments with respect to the intensity value obtained with the positive control (poly-L-lysine-coated plates). The significance of each binding assay was evaluated using the t test assuming unequal variances. P-values lower than 0.05 were considered statistically significant.

Mentions: The similarity of the MUC4-NIDO domain with the G1/NIDO domain of endogenous nidogen protein indicates that MUC4-NIDO domain may interact with nidogen-G1 domain interacting proteins. In this regard, a previous study has shown an interaction between fibulin-2 and the nidogen-G1 domain thus proving that fibulin-2 is one of the interacting proteins for the nidogen protein (Ries et al. 2001). The immunohistofluorescence analysis showed the colocalization of MUC4 and fibulin-2 in the exocrine glands of the adenocarcinoma tissues. However, both the proteins' expression was absent in the endocrine glands of the same tissue section (Figure 5A). Further, to test the possible interaction between the MUC4-NIDO domain with fibulin-2, we carried out a cell adhesion assay on the surface of fibulin-2 coated plates. We observed a significantly higher number of MiaPaCa-MUC4 cells binding to the fibulin-2 coated surface than MiaPaCa-MUC4-NIDOΔ or MiaPaCa-Vec cells (Figure 5B).


Role of MUC4-NIDO domain in the MUC4-mediated metastasis of pancreatic cancer cells.

Senapati S, Gnanapragassam VS, Moniaux N, Momi N, Batra SK - Oncogene (2011)

MUC4 NIDO domain interactions with Fibulin-2 in vitro, and colocalisation in pancreatic tissue(A) Immunohistofluorescence analysis was carried out on formalin-fixed and paraffin-embedded pancreatic tumor tissue sections after rehydration and antigen-retrieval. Digital merging of the immunostained tissue section clearly showed the colocalization of MUC4 (green) and fibulin-2 (red) in an adenocarcinoma lesion. Absence of MUC4 and fibulin-2 staining in the endocrine glands of the same tissue section acted as a negative control. (B) Binding Assay Cells (25 × 103) were seeded in 96-well plate coated with fibulin-2. Wells coated with bovine serum albumin and poly-L-lysine served as negative and positive controls, respectively. A total of 100 μl of the cell suspension was seeded in triplicates onto the fibulin-2-coated 96 well plates and incubated for 1 h at 37°C. Next the adhered cells were labeled with DIO dye for 1 h at 37°C. The fluorescence of the samples was measured by using the fluorescence plate reader at an excitation wavelength of 484 nm and an emission wavelength of 501 nm. The fluorescence intensity obtained in the negative control (BSA) was subtracted from the values from fibulin-2 coated plates. A relative fluorescence (RFU) was calculated for all treatments with respect to the intensity value obtained with the positive control (poly-L-lysine-coated plates). The significance of each binding assay was evaluated using the t test assuming unequal variances. P-values lower than 0.05 were considered statistically significant.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298579&req=5

Figure 5: MUC4 NIDO domain interactions with Fibulin-2 in vitro, and colocalisation in pancreatic tissue(A) Immunohistofluorescence analysis was carried out on formalin-fixed and paraffin-embedded pancreatic tumor tissue sections after rehydration and antigen-retrieval. Digital merging of the immunostained tissue section clearly showed the colocalization of MUC4 (green) and fibulin-2 (red) in an adenocarcinoma lesion. Absence of MUC4 and fibulin-2 staining in the endocrine glands of the same tissue section acted as a negative control. (B) Binding Assay Cells (25 × 103) were seeded in 96-well plate coated with fibulin-2. Wells coated with bovine serum albumin and poly-L-lysine served as negative and positive controls, respectively. A total of 100 μl of the cell suspension was seeded in triplicates onto the fibulin-2-coated 96 well plates and incubated for 1 h at 37°C. Next the adhered cells were labeled with DIO dye for 1 h at 37°C. The fluorescence of the samples was measured by using the fluorescence plate reader at an excitation wavelength of 484 nm and an emission wavelength of 501 nm. The fluorescence intensity obtained in the negative control (BSA) was subtracted from the values from fibulin-2 coated plates. A relative fluorescence (RFU) was calculated for all treatments with respect to the intensity value obtained with the positive control (poly-L-lysine-coated plates). The significance of each binding assay was evaluated using the t test assuming unequal variances. P-values lower than 0.05 were considered statistically significant.
Mentions: The similarity of the MUC4-NIDO domain with the G1/NIDO domain of endogenous nidogen protein indicates that MUC4-NIDO domain may interact with nidogen-G1 domain interacting proteins. In this regard, a previous study has shown an interaction between fibulin-2 and the nidogen-G1 domain thus proving that fibulin-2 is one of the interacting proteins for the nidogen protein (Ries et al. 2001). The immunohistofluorescence analysis showed the colocalization of MUC4 and fibulin-2 in the exocrine glands of the adenocarcinoma tissues. However, both the proteins' expression was absent in the endocrine glands of the same tissue section (Figure 5A). Further, to test the possible interaction between the MUC4-NIDO domain with fibulin-2, we carried out a cell adhesion assay on the surface of fibulin-2 coated plates. We observed a significantly higher number of MiaPaCa-MUC4 cells binding to the fibulin-2 coated surface than MiaPaCa-MUC4-NIDOΔ or MiaPaCa-Vec cells (Figure 5B).

Bottom Line: The in vitro studies demonstrated an enhanced invasiveness of MiaPaCa cells expressing MUC4 (MiaPaCa-MUC4) compared with vector-transfected cells (MiaPaCa-Vec; P=0.003) or cells expressing MUC4 without the NIDO domain (MiaPaCa-MUC4-NIDO(Δ); P=0.03).However, the absence of NIDO-domain has no significant role on cell growth and motility (P=0.93).Additionally, a reduced binding (P=0.0004) of MiaPaCa-MUC4-NIDO(Δ) cells to the fibulin-2 coated plates compared with MiaPaCa-MUC4 cells indicated a possible interaction between the MUC4-NIDO domain and fibulin-2, a nidogen-interacting protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.

ABSTRACT
MUC4 is a large transmembrane type I glycoprotein that is overexpressed in pancreatic cancer (PC) and has been shown to be associated with its progression and metastasis. However, the exact cellular and molecular mechanism(s) through which MUC4 promotes metastasis of PC cells has been sparsely studied. Here we showed that the nidogen-like (NIDO) domain of MUC4, which is similar to the G1-domain present in the nidogen or entactin (an extracellular matrix protein), contributes to the protein-protein interaction property of MUC4. By this interaction, MUC4 promotes breaching of basement membrane (BM) integrity, and spreading of cancer cells. These observations are corroborated with the data from our study using an engineered MUC4 protein without the NIDO domain, which was ectopically expressed in the MiaPaCa PC cells, lacking endogenous MUC4 and nidogen protein. The in vitro studies demonstrated an enhanced invasiveness of MiaPaCa cells expressing MUC4 (MiaPaCa-MUC4) compared with vector-transfected cells (MiaPaCa-Vec; P=0.003) or cells expressing MUC4 without the NIDO domain (MiaPaCa-MUC4-NIDO(Δ); P=0.03). However, the absence of NIDO-domain has no significant role on cell growth and motility (P=0.93). In the in vivo studies, all the mice orthotopically implanted with MiPaCa-MUC4 cells developed metastasis to the liver as compared with MiaPaCa-Vec or the MiaPaCa-MUC4-NIDO(Δ) group, hence, supporting our in vitro observations. Additionally, a reduced binding (P=0.0004) of MiaPaCa-MUC4-NIDO(Δ) cells to the fibulin-2 coated plates compared with MiaPaCa-MUC4 cells indicated a possible interaction between the MUC4-NIDO domain and fibulin-2, a nidogen-interacting protein. Furthermore, in PC tissue samples, MUC4 colocalized with the fibulin-2 present in the BM. Altogether, our findings demonstrate that the MUC4-NIDO domain significantly contributes to the MUC4-mediated metastasis of PC cells. This may be partly due to the interaction between the MUC4-NIDO domain and fibulin-2.

Show MeSH
Related in: MedlinePlus