Implications of increased tissue transglutaminase (TG2) expression in drug-resistant breast cancer (MCF-7) cells

Abstract

The development of resistance to chemotherapeutic drugs is a major obstacle to the successful treatment of breast cancer. Ways to block or overcome this resistance are objects of intense research. We have previously shown that cancer cells selected for resistance against chemotherapeutic drugs or isolated from metastatic tumor sites have high levels of a calcium-dependent protein crosslinking enzyme, tissue transglutaminase (TG2) but no direct link between TG2 and resistance was established. As TG2 can associate with the b members of the integrin family of proteins, we hypothesized that TG2 promotes cell survival signaling pathways by activating integrins on the surface of these cells. To test this hypothesis, we studied the expression of TG2 and its interaction with various integrins in drug-resistant MCF-7 breast cancer cells. TG2 closely associated with b1 and b5 integrins on the surface of drug-resistant MCF-7 (MCF-7/Dox and MCF-7/RT) cells. The incubation of TG2-expressing drug-resistant MCF-7 cells on fibronectin (Fn)-coated surfaces strongly activated focal adhesion kinase, an event that leads to the activation of several downstream signaling pathways and, in turn, can confer apoptosisresistant phenotype to cancer cells. The role of TG2 in Fnmediated cell attachment, cell growth, and cell survival functions was further analysed by small interfering RNA (siRNA) approach. Inhibition of TG2 by siRNA-inhibited Fn-mediated cell attachment and cell survival functions in drug-resistant MCF-7 cells. We conclude that the expression of TG2 in breast cancer cells contributes to the development of the drug-resistance phenotype by promoting interaction between integrins and Fn.

Tissue transglutaminase expression promotes cell attachment, invasion and survival in breast cancer cells

“…Moreover, ectopic expression of TG2 in MDA-231/cl.9 cells rendered these cells invasive and adherent on Fn-coated surfaces (Figure 8), thus further supporting a role for TG2 in these functions. We earlier reported a similar increase in TG2 expression in drug-resistant breast cancer cells (Chen et al, 2002(Chen et al, , 2004 and the association of TG2 with b-integrins in these cells (Herman et al, 2006). On the basis of these observations and in view of the highbinding affinity of TG2 for Fn (Jeong et al, 1995;Hang et al, 2005), we anticipated that TG2 expression would promote the stable interaction of integrins with Fn, the major matrix ligand in the ECM.…”

“…TG2 colocalized with integrin b1 (Figure 5c) and integrin b5 ( Figure 5d) in WT MDA-MB231 and MDA231/cl.16 cells, as suggested by the yellow fluorescence in the merged images. We next determined the involvement of TG2 in Fnmediated cell attachment, survival and invasion by using an siRNA approach (Herman et al, 2006). Transfection of MDA231/cl.16 cells with control siRNA (scrambled) had no appreciable effect on the TG2 levels.…”

“…Notably, the inhibition of TG2 protein by siRNA rendered the cells sensitive to apoptosis under serum-free conditions (Figure 7b). Similarly, inhibition of TG2 expression by siRNA or an antisense approach has been shown to reverse the sensitivity of drug-resistant breast cancer MCF-7 (Herman et al, 2006) and of lung cancer PC-14 cells (Han and Park, 1999) to chemotherapeutic drugs. Moreover, the culture of TG2-positive cells on Fn-coated surfaces has been shown to induce strong activation of the focal adhesion kinase Herman et al, 2006), an upstream event that leads to the activation of various downstream antiapoptotic and cell survival signaling pathways (Sonoda et al, 2000; McLean et al, 2005).…”

“…In some experiments, cells were transfected with TG2-specific (siRNA1 and siRNA2) or control (scrambled) siRNA as described earlier (Herman et al, 2006). After 48 h of TG2 expression and metastasis LS Mangala et al transfection, cells were harvested with 2 mM EDTA, washed and assayed for TG2 activity and levels or were plated in quadruplicate in each well of 96-well plates that had been precoated with Fn or BSA.…”

“…After washing with PBS, cells were incubated with a fluorescein isothiocyanate-conjugated antimouse IgG secondary antibody (0.2 mg/100 ml) and analysed Immunoprecipitation Four hundred micrograms of total cell lysate protein was used for the immunoprecipitation of integrins b1, b4 and b5 as described (Herman et al, 2006). Briefly, after preclearing and incubation with an appropriate antibody overnight, antigenantibody complexes were removed by incubation with antimouse IgG or anti-rabbit IgG for 1 h at 41C, followed by incubation with a G-protein sepharose beads (1-3 h at 41C).…”