CEACAM6 is a determinant of pancreatic adenocarcinoma cellular invasiveness - PubMed (original) (raw)

CEACAM6 is a determinant of pancreatic adenocarcinoma cellular invasiveness

M S Duxbury et al. Br J Cancer. 2004.

Abstract

Pancreatic adenocarcinoma is among the most aggressively invasive malignancies. The immunoglobulin superfamily member carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is emerging as an important determinant of the malignant phenotype in a range of cancers. We sought to define the role of CEACAM6 in pancreatic adenocarcinoma cellular invasiveness. CEACAM6 was stably overexpressed in Capan2 cells, which inherently express low levels of CEACAM6. Retrovirally mediated RNA interference was used to silence CEACAM6 expression in BxPC3 cells, which inherently overexpress CEACAM6. Cellular invasiveness was quantified using a modified Boyden chamber assay. Overexpression of CEACAM6 increased Capan2 cellular invasiveness, whereas CEACAM6 knockdown attenuated BxPC3 invasiveness. A role for the c-Src tyrosine kinase in mediating CEACAM6-dependent invasiveness was defined using constitutively active and dominant-negative c-Src expression constructs. c-Src-dependent modulation of matrix metalloproteinase-9 activity contributes significantly to the increased cellular invasiveness induced by CEACAM6 overexpression. Levels of CEACAM6 expression can modulate pancreatic adenocarcinoma cellular invasiveness in a c-Src-dependent manner. This pathway warrants further investigation as a target for therapy.

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Figures

Figure 1

(A) Stable overexpression of CEACAM6 was confirmed in two Capan2-derived transfectants, pIRES-CEACAM6.1 and pIRES-CEACAM6.2, by Western blotting. Representative example with mean densitometric values (±s.d.) from triplicate blots. *P<0.05 vs pIRES (empty vector) transfectants. (B) Cellular invasiveness in the Matrigel Boyden chamber assay was significantly increased in both pIRES-CEACAM6.1 and pIRES-CEACAM6.2 transfectants. Values are means (±s.d.) from triplicate experiments. *P<0.05 vs pIRES transfectants.

Figure 2

Stable suppression of CEACAM6 expression was demonstrated in BxPC3 psiCEACAM6 transfectants, by Western blot analysis. CEA expression did not significantly differ among the transfectants. Representative example with mean densitometric values (±s.d.) from triplicate blots. *P<0.05 vs both psiNeo (empty vector) and psiControl transfectants.

Figure 3

(A) Cellular invasiveness of psiCEACAM6 transfectants in the Matrigel Boyden chamber assay was significantly reduced, relative to psiNeo and psiControl transfectants. Values are means (±s.d.) from triplicate experiments. *P<0.05 vs both psiNeo and psiControl transfectants. (B) Cellular proliferation of psiCEACAM6 transfectants, quantified by MTT assay, did not significantly differ from that of psiNeo or psiControl transfectants. Values are means (±s.d.) from triplicate experiments.

Figure 4

Both pIRES-CEACAM6.1 and pIRES-CEACAM6.2 Capan2-derived transfectants exhibited increased c-Src kinase activity, relative to pIRES transfectants. Conversely, suppression of CEACAM6 expression was associated with decreased c-Src kinase activity in BxPC3-derived psiCEACAM6 transfectants. Values are means (±s.d.) from triplicate experiments. *P<0.05 vs pIRES transfectants. †P<0.05 vs psiControl and psiNeo transfectants.

Figure 5

(A) MMP-9 activities were significantly increased in pIRES-CEACAM6.1 and pIRES-CEACAM6.2 transfectants, relative to pIRES transfectants. psiCEACAM6 transfectants exhibited significantly lower MMP-9 activity than both psiNeo and psiControl transfectants. Mean (±s.d.) from triplicate experiments. *P<0.05 vs respective control transfectants. (B) Cellular invasiveness of pIRES-CEACAM6.1 and pIRES-CEACAM6.2 transfectants was significantly attenuated by exposure to anti-MMP-9 monoclonal antibody, but not isotype-matched irrelevant IgG. Mean (±s.d.) from triplicate experiments. *P<0.05 vs control IgG. Overexpression of dominant-negative Src (DN Src) significantly decreased cellular invasiveness (C) and MMP-9 activities (D) of pIRES-CEACAM6.1 and pIRES-CEACAM6.2 transfectants. Constitutively active c-Src induced a significant recovery of MMP-9 activity (C) and cellular invasiveness (D) in psiCEACAM6 transfectants. Values are means (±s.d.) from triplicate experiments. *P<0.05 vs pUSE empty vector.

Figure 6

(A) Sustained knockdown of CEACAM6 expression was confirmed in the excised tumours by Western blot analysis. A representative blot is shown. (B) MMP-9 activities in lysates obtained from psiCEACAM6 transfectants were significantly lower than those of psiControl-derived tumours. Mean values (±s.d.). *P<0.05 vs psiControl transfectant tumours.

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CEACAM6 is a determinant of pancreatic adenocarcinoma cellular invasiveness - PubMed (original) (raw)