Uncoupled responses of Smad4-deficient cancer cells to TNFalpha result in secretion of monomeric laminin-gamma2

Abstract

Background: Functional loss of the tumor suppressor Smad4 is involved in pancreatic and colorectal carcinogenesis and has been associated with the acquisition of invasiveness. We have previously demonstrated that the heterotrimeric basement membrane protein laminin-332 is a Smad4 target. Namely, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-332; its loss is thus implicated in the reduced or discontinuous deposition of the heterotrimeric basement membrane molecule as evident in carcinomas. Uncoupled expression of laminin genes, on the other hand, namely overexpression of the laminin-gamma2 chain is an impressive marker at invasive edges of carcinomas where tumor cells are maximally exposed to signals from stromal cell types like macrophages. As Smad4 is characterized as an integrator of multiple extracellular stimuli in a strongly contextual manner, we asked if loss of Smad4 may also be involved in uncoupled expression of laminin genes in response to altered environmental stimuli. Here, we address Smad4 dependent effects of the prominent inflammatory cytokine TNFalpha on tumor cells.

Results: Smad4-reconstituted colon carcinoma cells like adenoma cells respond to TNFalpha with an increased expression of all three chains encoding laminin-332; coincubation with TGFbeta and TNFalpha leads to synergistic induction and to the secretion of large amounts of the heterotrimer. In contrast, in Smad4-deficient cells TNFalpha can induce expression of the gamma2 and beta3 but not the alpha3 chain. Surprisingly, this uncoupled induction of laminin-332 chains in Smad4-negative cells rather than causing intracellular accumulation is followed by the release of gamma2 into the medium, either in a monomeric form or in complexes with as yet unknown proteins. Soluble gamma2 is associated with increased cell migration.

Conclusions: Loss of Smad4 may lead to uncoupled induction of laminin-gamma2 in response to TNFalpha and may therefore represent one of the mechanisms which underlie accumulation of laminin-gamma2 at the invasive margin of a tumor. The finding, that gamma2 is secreted from tumor cells in significant amounts and is associated with increased cell migration may pave the way for further investigation to better understand its functional relevance for tumor progression.

Figure 1

Synergistic induction of laminin-332 in human adenoma cells in response to inflammatory cytokines TGFβ and TNFα. (A) Western blot analysis of heterotrimeric laminin-332 expressed by LT97 colorectal adenoma cells. Proteins (8 μg/lane) prepared from serum-free conditioned media from LT97 cells treated with recombinant TGFβ and TNFα for 48 h as indicated were separated on 3-8% tris-acetate gradient gels (Invitrogen) under non-reducing conditions. The blot was probed with a laminin-γ2-specific antibody (polyclonal antibody 2140, PS-A) and reprobed with a transferrin-specific antibody used as a loading control. The bars indicate the relative signal strength normalized for transferrin. Note that the Odyssey detection system (LI-COR) allows for a direct digital quantification of signals. Similar results were obtained in > three experiments. The same signals, although with less sensitivity, were obtained using a commercial antibody (MAB-19562, Chemicon). (B) Northern blot analyses of the LAMA3, LAMB3 and LAMC2 genes prepared with RNAs from LT97 cells treated with cytokines for 24 h. Quantification of mRNA levels was done by phosphorimage analysis and signal strengths normalized with GAPDH. Similar results were obtained in three experiments.

Figure 2

Synergistic effect of TGFβ and TNFα on the secretion of heterotrimeric laminin-332 by Smad4-reconstituted human colorectal cancer cells and uncoupled responses of Smad4-deficient cells. (A and B) Western blot analysis of proteins from conditioned media (12 μg of protein per lane) produced by SW480 (A) and SW620 (B) cells as described in figure 1. (C) Northern Blot analysis with RNAs from SW620 cells treated with cytokines for 4 h. Shown in each bar is the mean +/- standard error (n = 3). The additional file 2 provides additional data for SW620 cells treated with cytokines for 24 hours and data for SW480 cells treated with cytokines for 4 and 24 hours.

Figure 3

TNFα induced secretion of monomeric laminin-γ2 by Smad4-deficient colorectal cancer cells. (A) Western blot analysis of proteins from conditioned media produced by SW620 cells in response to treatment with recombinant TGFβ and TNFα for 48 h under non-reducing conditions. Proteins (16 μg/lane) were separated by SDS-PAGE on 8% polyacrylamide gels, blotted and probed with a laminin-γ2-specific antibody (2140). (B) A set of each, three independent Smad4-deficient and Smad4-reexpressing SW620 cell clones were treated with TNFα and conditioned media analysed like in (A). (C) Protein samples corresponding to those used in (A) were analyzed under reducing conditions.

Figure 4

Mass spectrometry-based confirmation of secreted laminin-γ2. (A) Preparative gel electrophoresis of conditioned media from SW620 cells (Smad4-deficient) treated with TNFα. An 8% SDS-PAGE was performed with preparative amounts of protein (32 μg) in the middle lane and analytical amounts of protein (16 μg) in the left and right lanes under non-reducing conditions. The left and right lanes were used for Western blotting with a laminin-γ2-specific antibody. Four small slices (1-2 mm) corresponding to the Western blot signals were cut from the preparative gel lane and proteins analysed by mass spectrometry. (B) Amino acid sequence of laminin-γ2. The peptides identified by mass spectrometry in gel bands 1-4 are indicated.

Figure 5

Induced release of monomeric laminin-γ2 upon transient laminin-α3 knockdown in SW620 cells and its impact on cell migration. (A) Northern blot analysis of LAMA3 knockdown in SW620 cells. RNAs were prepared from SW620 cells transiently transfected with LAMA3 siRNA and incubated with TNFα for 24 h. Quantification of LAMA3 messages normalized for GAPDH is indicated. (B) Western blot analysis of laminin-γ2 expression upon transient knockdown of LAMA3. SW620 cells transiently transfected with LAMA3 siRNA or non-targeting siRNA were shifted to serum-free cultures 48 h after transfection and were incubated with TNFα for another 48 h. Proteins from conditioned media (16 μg/lane) were probed with a laminin-γ2-specific antibody (2140). Quantification of the monomer and of the heterotrimer normalized for transferrin is indicated. (C) Migration of SW620 cells as analyzed in a transwell migration assay. SW620 cells transfected with LAMA3 siRNA or non-targeting siRNA were plated in a transwell chamber 24 h after transfection. TNFα was added one day later. Migrating cells were quantified after three days using Cell Titer Glo assay (Promega). Bars show the mean value of four experiments with the standard error of the mean. Statistical analysis was carried out by t test (one-tailed, GraphPad Prism 4.00).

Figure 6

Analysis of regulatory sites involved in TNFα responses of the LAMA3 promoter by transient transfection of promoter-reporter constructs. Normalized promoter activities of LAMA3 wild-type and mutated promoter constructs. SW480 cells were plated in 96-well plates and transfected with the indicated promoter constructs using the Dual-Luciferase-Reporter Assay System (Promega). An NF-κB site is involved in promoter responses in Smad4-positive but not in Smad4-negative cells. An AP1 site is involved in TNFα responsiveness in a Smad4-independent manner. Bars show the mean value of three approaches with the standard error of the mean. Statistical analysis was carried out by t test (one-tailed, GraphPad Prism 4.00). *P < 0.05, **P < 0.01