p38MAPK induces cell surface alpha4 integrin downregulation to facilitate erbB-2-mediated invasion - PubMed (original) (raw)

p38MAPK induces cell surface alpha4 integrin downregulation to facilitate erbB-2-mediated invasion

Kathleen M Woods Ignatoski et al. Neoplasia. 2003 Mar-Apr.

Abstract

We have previously shown that human breast cancer cells that overexpress erbB-2 are growth factor-independent. In order to test the contribution of erbB-2 to this and other transformed phenotypes without the genetic instability of cancer cells, erbB-2 was overexpressed in human mammary epithelial (HME) cells. ErbB-2-overexpressing HME cells exhibit several transformed phenotypes including cell surface alpha(4) integrin downregulation and invasiveness. We formulated a model for invasiveness that depends on a cell's ability to downregulate alpha(4) integrin. As small G-proteins play a role in cytoskeleton remodeling and as this is a likely route for alpha(4) integrin trafficking, we investigated the role of small G-proteins and their downstream signals in mediating alpha(4) integrin downregulation and invasiveness using Rac 1. Dominant-negative Rac 1 blocked erbB-2-mediated invasion and reversed erbB-2-mediated alpha(4) integrin downregulation. In addition, constitutively active Rac 1 induced alpha(4) integrin downregulation and invasiveness. In erbB-2-overexpressing and in constitutively active Rac 1-expressing cells, a p38MAP kinase (p38MAPK) inhibitor blocked invasiveness and reversed alpha(4) integrin downregulation. These data suggest a model in which erbB-2 signaling activates Rac 1, which, in turn, activates p38MAPK, leading to the downregulation of alpha(4) integrin. These data strengthen the model where loss of alpha(4) integrin at the cell surface, leading to reduced alpha(4) integrin binding to plasma fibronectin, plays a role in erbB-2-mediated invasiveness.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Model for invasion. Normal HME cells express the Fn receptors α4β1 and α5β1 integrin on the cell surface and are not able to invade basement membrane substrates. α4β1 and α5β1 integrin bind plasma Fn. α5 integrin can bind the PHSRN sequence in module 9 of plasma Fn, whereas α4 recognizes plasma Fn C-terminal to the α5 recognition sites. We have shown that HME cells that are binding PHSRN-containing fragments without α4 binding to Fn are able to invade. HBC cells and erbB-2-overexpressing HME cells have lost the expression of α4 integrin on the cell surface and are able to invade basement membranes. Therefore, the interaction of plasma Fn with α5β1 integrin is essential for invasion, and the presence of α4β1 binding to plasma Fn blocks the Fn-induced invasion signals. Data we have published elsewhere indicate that α5β1 signaling goes through MMP-1 to mediate invasion.

Figure 2

Figure 2

Invasion induced by erbB-2 is inhibited by a variety of pathways. ErbB-2-overexpressing cells were incubated for 24 hours with 1 µM of the pan-erbB inhibitor CI-1033, 10 µM of the MEK inhibitor PD98059, 25 µM of the PI3K inhibitor LY294002, or 1 µM of the Src inhibitor PD173952, then subjected to SU-ECM invasion assays. Invasion percentages are relative to the invasion capacity of untreated erbB-2 cells. Each assay was performed in duplicate and repeated at least twice.

Figure 3

Figure 3

Rac 1 facilitates erbB-2-mediated invasion. H16N2 cells were infected with retroviral expression vectors containing Rac and PAK constructs. Proteins from whole cell lysates were separated on 10% SDS-PAGE and blotted to PVDF membrane. (A) To validate expression of Rac V12, immunoblots were prepared and probed with an anti-Rac 1 antibody. Endogenous and exogenous Rac 1 are indicated. (B) Constitutively active Rac 1 (Rac V12)-expressing cells, dominant negative Rac 1 (Rac N17)-expressing erbB-2 cells, or erbB-2-overexpressing cells containing the dominantly acting PAK construct (PAK 83–149) or a control construct (PAK 83–149/L107F) were subjected to SU-ECM invasion assays. Invasion percentages are relative to the invasion capacity of untreated erbB-2 cells. Each assay was performed in duplicate and repeated at least twice.

Figure 4

Figure 4

Invasiveness is dependent on p38MAPK. SUM-52PE human breast cancer cells, ErbB-2-overexpressing HME cells, and Rac V12-expressing cells were exposed for 24 hours to 10 µM SB202190, then were subjected to SU-ECM invasion assays. Invasion percentages are relative to the invasion capacity of untreated erbB-2 cells. Each assay was performed in duplicate and repeated at least twice.

Figure 5

Figure 5

Rac 1-mediated invasiveness is dependent on PI3K. (A) To confirm expression of exogenous PTEN in transduced cells, immunoblots were prepared and probed with anti-PTEN antibody. (B) SU-ECM invasion assay of Rac V12-expressing cells, Rac V12-expressing cells exposed to 25 µM LY294002, or Rac V12-expressing cells containing exogenous PTEN. Invasion percentages are relative to the invasion capacity of untreated Rac V12 cells. Each assay was performed in duplicate and repeated at least twice.

Similar articles

Cited by

References

    1. Sporn MB. The war against cancer. Lancet. 1997;347:1377–1381. - PubMed
    1. Berger MS, Locher GW, Saurer S, Gullick WJ, Waterfield MD, Groner B, Hynes NE. Correlation of c-erbB-2 gene amplification and protein expression in human breast carcinoma with nodal status and nuclear grading #132. Cancer Res. 1988;48:1238–1243. - PubMed
    1. Guerin M, Gabillot M, Mathieu MC, Travagli JP, Spielmann NA, Riou G. Structure and expression of c-erbB-2 and EGF receptor genes inflammatory and non-inflammatory breast cancer: prognostic significance. Int J Cancer. 1989;43:201–208. - PubMed
    1. Woods Ignatoski KM, LaPointe AJ, Radany EH, Ethier SP. ErbB-2 overexpression in human mammary epithelial cells confers growth factor independence. Endocrinology. 1999;140:3615–3622. - PubMed
    1. Woods Ignatoski KM, Maehama T, Markwart SM, Dixon JE, Livant DL, Ethier SP. ERBB-2 overexpression confers PI 3′-kinase-dependent invasion capacity on human mammary epithelial cells. Br J Cancer. 2000;82:666–674. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources