Purpose: Drug resistance and metastasis pose major impediments in the successful treatment of cancer. We previously reported that multidrug-resistant breast cancer cells exhibit high levels of tissue transglutaminase (TG2; EC 2.3.2.13). Because the drug-resistant and metastatic phenotypes are thought to share some common pathways, we sought to determine whether metastatic breast cancer cells express high levels of TG2. Experimental Design: The metastatic breast cancer cell line MDA-MB-231 and the sublines derived from it were tested for TG2 expression. Similarly, several sublines derived from an immortal but normal breast epithelial cell line, MCF10A, representing various stages in breast cancer progression were studied for TG2 expression. The primary and nodal tumor samples from 30 patients with breast cancer were also studied for TG2 expression. Results: The MDA-MB-231 cells expressed high basal levels of TG2. Two clones derived from this cell line, MDA231/cl.9 and MDA231/cl.16, showed a 10- to 15-fold difference in TG2 level. TG2-deficient MDA231/cl.9 cells exhibited higher sensitivity to doxorubicin and were less invasive than were the TG2-sufficient MDA231/cl.16 cells. The MCF10A-derived sublines had increased TG2 expression as they advanced from noninvasive to an invasive phenotype. Importantly, the metastatic lymph node tumors from patients with breast cancer showed significant higher levels of TG2 expression compared with the primary tumors from the same patients. Conclusions: TG2 expression is up-regulated in drug-resistant and metastatic breast cancer cells, and it can serve as a valuable prognostic marker for these phenotypes.

Recent observations that aberrant expression of tissue transglutaminase (TG2) promotes growth, survival, and metastasis of multiple tumor types is of great significance and could yield novel therapeutic targets for improved patient outcomes. To accomplish this, a clear understanding of how TG2 contributes to these phenotypes is essential. Using mammary epithelial cell lines (MCF10A, MCF12A, MCF7 and MCF7/RT) as a model system, we determined the impact of TG2 expression on cell growth, cell survival, invasion, and differentiation. Our results show that TG2 expression promotes drug resistance and invasive functions by inducing epithelial-mesenchymal transition (EMT). Thus, TG2 expression supported anchorage-independent growth of mammary epithelial cells in soft-agar, disrupted the apical-basal polarity, and resulted in disorganized acini structures when grown in 3D-culture. At molecular level, TG2 expression resulted in loss of E-cadherin and increased the expression of various transcriptional repressors (Snail1, Zeb1, Zeb2 and Twist1). Tumor growth factor-beta (TGF-β) failed to induce EMT in cells lacking TG2 expression, suggesting that TG2 is a downstream effector of TGF-β-induced EMT. Moreover, TG2 expression induced stem cell-like phenotype in mammary epithelial cells as revealed by enrichment of CD44⁺/CD24^-/low cell populations. Overall, our studies show that aberrant expression of TG2 is sufficient for inducing EMT in epithelial cells and establish a strong link between TG2 expression and progression of metastatic breast disease.

Background: Alterations in the extracellular matrix (ECM) can affect host-tumor interactions and tumor growth and metastasis. Tissue transglutaminase (TG2, EC 2.3.2.13), a calcium-dependent enzyme that catalyzes covalent cross-linking of proteins, can render the ECM highly stable and resistant to proteolytic degradation. So we determined whether TG2 expression in a tumor or nontumor (stroma) environment could affect the process of metastasis. Two hundred archived samples from patients with breast cancer were studied for the TG2 expression. Also, in an in vitro model the invasive behavior of MDA-MB-231 cells in the presence or absence of exogenous TG2 was determined. Results: Tumors associated with negative nodes showed significantly higher expression of TG2 in the stroma (P < 0.001). TG2 in the stroma was catalytically active, as revealed by the presence of isopeptide cross-links. Pretreatment of Matrigel with catalytically active TG2 resulted in strong inhibition of invasion of MDA-MB-231 cells through the Matrigel Transwell filters. Conclusion: TG2-induced alterations in the ECM could effectively inhibit the process of metastasis. Therefore, selective induction of catalytically active TG2 at the site of tumor may offer promising approach for limiting the metastasis.