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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.

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Macroscopic examinations of orthotopic pancreatic tumors and organs with metastatic lesions(A). Macroscopic appearance of primary pancreatic tumors from animals implanted with MiaPaCa-MUC4, MiaPaCa-MUC4-NIDOΔ and MiaPaCa-Vect cells. There was no difference in the gross appearance of tumors derived from MiaPaCa-MUC4 and MiaPaCa-MUC4-NIDOΔ groups. However, tumors of the MiaPaCa-Vect group were smaller than the other two groups. Further, metastasis to organs such as the diaphragm and mesentery was same in all the three groups excluding liver metastasis, which was observed only in animals injected with MiaPaCa-MUC4 cells. (B) Quantitative analysis showed a significantly smaller tumor generated from MiaPaCa-Vect cells than MiaPaCa-MUC4 and MiaPaCa-MUC4-NIDOΔ. Further analysis of metastatic lesions among the three groups showed no significant difference in the number of metastatic nodules that were detected in mesentery and the diaphragm. However, there was a significant difference (p=0.001) in the number of metastatic nodules present in the liver.
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Figure 3: Macroscopic examinations of orthotopic pancreatic tumors and organs with metastatic lesions(A). Macroscopic appearance of primary pancreatic tumors from animals implanted with MiaPaCa-MUC4, MiaPaCa-MUC4-NIDOΔ and MiaPaCa-Vect cells. There was no difference in the gross appearance of tumors derived from MiaPaCa-MUC4 and MiaPaCa-MUC4-NIDOΔ groups. However, tumors of the MiaPaCa-Vect group were smaller than the other two groups. Further, metastasis to organs such as the diaphragm and mesentery was same in all the three groups excluding liver metastasis, which was observed only in animals injected with MiaPaCa-MUC4 cells. (B) Quantitative analysis showed a significantly smaller tumor generated from MiaPaCa-Vect cells than MiaPaCa-MUC4 and MiaPaCa-MUC4-NIDOΔ. Further analysis of metastatic lesions among the three groups showed no significant difference in the number of metastatic nodules that were detected in mesentery and the diaphragm. However, there was a significant difference (p=0.001) in the number of metastatic nodules present in the liver.

Mentions: To investigate the role of the MUC4-NIDO domain in vivo, we carried out the orthotopic implantation of MiaPaCa cells expressing MUC4 or MUC4 without the NIDO domain. In parallel, we also injected vector transfected MiaPaCa cells as a control (MiaPaCa-Vec). For this experiment, two millions of MiaPaCa-derived cells were injected into the head of the pancreas, and animals were sacrificed after 65days of the cancer cells injection. During autopsy of these mice, we did not find a significant difference in the mean tumor weight between MiaPaCa-MUC4 and MiaPaCa- MUC4-NIDOΔ groups (Figure 3B). However, animals in both the groups have a significantly higher mean tumor weight than the vector control animals (Figure 3B; p<0.005). Importantly, we found that the only animal group that developed massive metastasis to the liver was the one implanted with MUC4 expressing MiaPaCa cells. In this particular group of animals, out of eight animals, seven animals had liver metastasis and the average number of metastatic nodules per animal was ∼3. Incidence of metastasis to other organs like spleen, mesentery, peritoneum and diaphragm was similar in all the three groups (Figure 3A, B, and Table 1). Furthermore, histopathological evaluation of primary and metastatic tumors obtained from MiaPaCa-MUC4 and MiaPaCa- MUC4-NIDOΔ groups did not show any gross difference in the pattern of local tissue invasiveness of the cancer cells (Figure 4).


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)

Macroscopic examinations of orthotopic pancreatic tumors and organs with metastatic lesions(A). Macroscopic appearance of primary pancreatic tumors from animals implanted with MiaPaCa-MUC4, MiaPaCa-MUC4-NIDOΔ and MiaPaCa-Vect cells. There was no difference in the gross appearance of tumors derived from MiaPaCa-MUC4 and MiaPaCa-MUC4-NIDOΔ groups. However, tumors of the MiaPaCa-Vect group were smaller than the other two groups. Further, metastasis to organs such as the diaphragm and mesentery was same in all the three groups excluding liver metastasis, which was observed only in animals injected with MiaPaCa-MUC4 cells. (B) Quantitative analysis showed a significantly smaller tumor generated from MiaPaCa-Vect cells than MiaPaCa-MUC4 and MiaPaCa-MUC4-NIDOΔ. Further analysis of metastatic lesions among the three groups showed no significant difference in the number of metastatic nodules that were detected in mesentery and the diaphragm. However, there was a significant difference (p=0.001) in the number of metastatic nodules present in the liver.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Macroscopic examinations of orthotopic pancreatic tumors and organs with metastatic lesions(A). Macroscopic appearance of primary pancreatic tumors from animals implanted with MiaPaCa-MUC4, MiaPaCa-MUC4-NIDOΔ and MiaPaCa-Vect cells. There was no difference in the gross appearance of tumors derived from MiaPaCa-MUC4 and MiaPaCa-MUC4-NIDOΔ groups. However, tumors of the MiaPaCa-Vect group were smaller than the other two groups. Further, metastasis to organs such as the diaphragm and mesentery was same in all the three groups excluding liver metastasis, which was observed only in animals injected with MiaPaCa-MUC4 cells. (B) Quantitative analysis showed a significantly smaller tumor generated from MiaPaCa-Vect cells than MiaPaCa-MUC4 and MiaPaCa-MUC4-NIDOΔ. Further analysis of metastatic lesions among the three groups showed no significant difference in the number of metastatic nodules that were detected in mesentery and the diaphragm. However, there was a significant difference (p=0.001) in the number of metastatic nodules present in the liver.
Mentions: To investigate the role of the MUC4-NIDO domain in vivo, we carried out the orthotopic implantation of MiaPaCa cells expressing MUC4 or MUC4 without the NIDO domain. In parallel, we also injected vector transfected MiaPaCa cells as a control (MiaPaCa-Vec). For this experiment, two millions of MiaPaCa-derived cells were injected into the head of the pancreas, and animals were sacrificed after 65days of the cancer cells injection. During autopsy of these mice, we did not find a significant difference in the mean tumor weight between MiaPaCa-MUC4 and MiaPaCa- MUC4-NIDOΔ groups (Figure 3B). However, animals in both the groups have a significantly higher mean tumor weight than the vector control animals (Figure 3B; p<0.005). Importantly, we found that the only animal group that developed massive metastasis to the liver was the one implanted with MUC4 expressing MiaPaCa cells. In this particular group of animals, out of eight animals, seven animals had liver metastasis and the average number of metastatic nodules per animal was ∼3. Incidence of metastasis to other organs like spleen, mesentery, peritoneum and diaphragm was similar in all the three groups (Figure 3A, B, and Table 1). Furthermore, histopathological evaluation of primary and metastatic tumors obtained from MiaPaCa-MUC4 and MiaPaCa- MUC4-NIDOΔ groups did not show any gross difference in the pattern of local tissue invasiveness of the cancer cells (Figure 4).

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