EGF potentiated oncogenesis requires a tissue transglutaminase-dependent signaling pathway leading to Src activation

Abstract

EGF receptor (EGFR) signaling in human cancers elicits changes in protein-expression patterns that are crucial for potentiating tumor growth. Identifying those proteins with expression regulated by the EGFR and determining how they contribute0 to malignancy is fundamental for the development of more effective strategies to treat cancer. Here, we show that tissue transglutaminase (tTG) is one such protein. EGF up-regulates tTG expression in human breast-cancer cells, and knock-downs of tTG or the treatment of breast cancer cells with a tTG inhibitor blocks their EGF-stimulated anchorage-independent growth. We further show that the combined actions of Ras and Cdc42, leading to the activation of PI 3-kinase and NFκB, provide a mechanism by which EGF can up-regulate tTG in breast-cancer cells. Moreover, overexpression of wild-type tTG, but not its transamidation-defective counterpart, fully mimics the growth advantages afforded by EGF to these cancer cells. Surprisingly, the tTG-promoted growth of breast-cancer cells is dependent on its ability to activate the Src tyrosine kinase as an outcome of a complex formed between tTG and the breast-cancer marker and intermediate filament protein keratin-19. These findings identify tTG as a key participant in an EGFR/Src-signaling pathway in breast-cancer cells and a potential target for inhibiting EGFR-promoted tumor progression.

The EGF receptor (EGFR) is expressed on the surface of various types of human cancer cells where the ligand-dependent induction of its kinase activity triggers signaling pathways that promote cancer-cell growth, motility, and chemo-resistance (1). Moreover, the EGFR or one of its ligands is often found overexpressed or mutated in tumors, and these events are linked to enhanced tumorigenicity (2, 3). The close connection between excessive EGFR-signaling activities and cancer progression has led to the development of EGFR-targeted therapeutic strategies that are currently being used to treat patients with lung, colon, and pancreatic cancer (1, 4). However, many of these patients either fail to respond or develop resistance to these treatments, suggesting that alternative strategies that target EGFR-signaling activities are needed and could be therapeutically beneficial in these cases (5).

Determining the molecular mechanisms that underlie the ability of the EGFR to stimulate malignant transformation represents a crucial step in the development of more effective and/or alternative cancer treatments. Indeed, several of the signaling proteins that have been shown to function downstream of the EGFR, including Ras, PI3K, and Src, have been extensively investigated for their roles in mediating oncogenic transformation, and they are now considered as viable targets for intervention against various types of human cancer (3, 6, 7). In addition, the EGFR regulates the expression of a number of proteins likely to contribute to cell-growth control and malignant transformation, and these proteins offer an additional group of potential therapeutic targets. Here, we show that tissue transglutaminase (tTG), which is capable of GTP-binding/GTPase activity and acyl transferase activity (8, 9), represents one such protein whose expression is strongly stimulated by EGF in human breast-cancer cells. We go on to show how tTG expression is up-regulated by EGFR-signaling activities and that it plays an essential role in EGFR-driven oncogenic transformation.

Results

tTG Mediates the Growth-Promoting Effects of EGF in Human Breast-Cancer Cells.

The human breast-cancer cell line SKBR3, when grown in medium containing EGF, shows enhanced proliferation compared with control cells (Fig. S1A) and an increased ability to form colonies in soft agar (Fig. S1 B and C), an in vitro measure of tumorgenicity. Efforts to identify proteins in these breast-cancer cells, whose expression was up-regulated by EGF and therefore, potentially could contribute to its growth-promoting activity, led us to tTG, a dual-function enzyme that couples an ability to bind and hydrolyze GTP with a transamidation activity that cross-links proteins through glutamine-lysine or glutamine-polyamine linkages. Whereas there have been suggestions that chronic tTG expression and/or activation associated with some human cancers is linked to their malignant transformation (10 –16), an understanding of how tTG is regulated in cancer cells and whether or not there is a functional relationship between this protein and EGF-promoted tumor growth has been lacking. Fig. 1A Inset shows that EGF induced a strong up-regulation of tTG expression in SKBR3 cells compared with the nearly undetectable amounts in untreated cells. Additionally, it stimulated its enzymatic activity as assayed by the incorporation of biotinylated pentylamine (BPA) into lysate proteins (Fig. 1A, graph). Similarly, EGF caused the up-regulation and activation of tTG in other breast-cancer cell lines such as BT20 cells (Fig. 1B Inset, graph).

Fig. 1.

tTG is required for the EGF-stimulated growth of human breast-cancer cells. (A and B) Serum-starved SKBR3 cells (A) and BT20 cells (B) were treated without or with EGF for 2 days and then lysed. The cell extracts were immunoblotted with tTG and actin antibodies (Insets) and assayed for transamidation activity by the incorporation of BPA into lysate proteins. The results from three separate assays were quantified using ImageJ software, averaged together, and graphed (graphs). (C and D) Two sets each of SKBR3 cells (C) or BT20 cells (D) transfected with control siRNA or tTG siRNAs (denoted tTG siRNA-1 or tTG siRNA-2) or incubated without or with MDC were stimulated without or with EGF as indicated. One set of cells was lysed and immunoblotted with tTG and actin antibodies (Insets). Anchorage-independent growth assays were performed on the second set of cells. The results from three separate assays were averaged together and graphed (graphs). (E) Equal numbers of SKBR3 cells transfected with control siRNA or tTG siRNAs or incubated without or with MDC were plated and then maintained in medium containing 2% FBS supplemented without or with EGF. The cell cultures were counted at the indicated times, and their growth rates were recorded.

We further established a link between growth-factor signaling and tTG in human breast-cancer cells by providing two additional pieces of evidence. First, we found that heregulin, a ligand that activates the EGFR family member ErbB2, which has been implicated in breast-cancer progression (1), was nearly as effective as EGF at inducing tTG expression and activation in SKBR3 cells (Fig. S2). Second, immunohistochemical analysis of a human breast-cancer tissue array shows that the expression levels of both the EGFR and ErbB2 correlate significantly to tTG expression (Fig. S3 A–C). Taken together, these findings suggest that tTG may be a critical mediator of EGF- and heregulin-promoted oncogenic processes.

We then asked whether or not knocking-down tTG expression by siRNA (Fig. 1 C and D Insets) or the addition of monodansylcadaverine (MDC), an inhibitor of tTG-catalyzed transamidation, affected the growth-promoting characteristics of EGF. Soft-agar assays showed that the EGF-stimulated colony formation of SKBR3 and BT20 cells was reduced to essentially basal levels (i.e., the number of colonies that form in the absence of EGF stimulation) by either the introduction of siRNAs targeting tTG or by treatment with MDC (Fig. 1 C and D, graphs). In addition, as shown in Fig. 1E, knock-downs of tTG or treatment with MDC also inhibited the ability of EGF to stimulate the growth of SKBR3 cells in monolayer.

Coactivation of the Ras- and Cdc42-Signaling Cascades Promotes tTG Expression.

To determine how EGF promotes the up-regulation of tTG expression, we examined several signaling proteins known to act downstream of the EGFR, including the small GTPases Ras, Rac1, Cdc42, and RhoA as well as PI3K and focal adhesion kinase (FAK) (17). Fig. 2A shows the results obtained when we introduced activated forms of these different GTPases, PI3K [myristoylated (M)-PI3K] or wild-type FAK, into SKBR3 cells and assayed for changes in the expression levels of tTG. Despite expressing considerable amounts of each protein (Fig. 2A Middle), none was capable of enhancing tTG expression (Fig. 2A Top). This suggested that the ability of EGF to induce tTG expression is likely not mediated by a single signaling cascade, but rather, it results from the combined activation of two or more pathways. In fact, we found that when different combinations of activated Ras and Rho GTPases (e.g., Cdc42) were expressed in SKBR3 cells, there was a marked up-regulation of tTG expression that fully mimicked the actions of EGF (Fig. 2B). Whereas combinations of activated Ras and Cdc42 or Ras and Rac1 seemed to be most effective at restoring tTG expression, different combinations of Rho GTPases in the absence of activated Ras (e.g., Cdc42 and RhoA) or the expression of oncogenic Dbl, a guanine nucleotide-exchange factor for Cdc42 and RhoA (18), will at least partially mirror the actions of EGF in up-regulating tTG (Fig. S4).

Fig. 2.

Ras- and Cdc42-dependent signaling pathways stimulate tTG expression. (A) Extracts of serum-starved SKBR3 cells ectopically expressing either vector (treated without or with EGF) or one of the activated forms of the indicated signaling proteins for 2 days were immunoblotted with tTG, HA, and actin antibodies as indicated. (B) Serum-deprived cells expressing the vector (treated without or with EGF) or various combinations of the activated forms of Ras (Ras G12V) and Cdc42 (Cdc42 F28L) were lysed and then immunoblotted with the indicated antibodies. (C) SKBR3 cells cultured in serum-free medium supplemented without or with EGF, plus either LY294002, PD98059, or BAY 11-7082 for 2 days, were lysed, and the extracts were immunoblotted with tTG and actin antibodies. The level of tTG expression detected in each sample was quantified using ImageJ software and listed below the tTG blot (fold tTG expression). (D) SKBR3 cells expressing Ras G12V and/or Cdc42 F28L were incubated without or with either LY294002 or BAY 11-7082 for 2 days and then were lysed. The extracts were immunoblotted with the indicated antibodies.

The ability of the activated Ras and Rho proteins to work together to up-regulate tTG expression led to the question of what might be functioning downstream from these GTPases. One of the best known signaling effectors for activated Ras is ERK, and so, we examined how blocking ERK activity using the inhibitor PD98059 affected EGF-stimulated tTG expression. The results in Fig. 2C show that the EGF-stimulated expression of tTG in SKBR3 cells was unaffected under conditions where ERK activity was inhibited. However, another well-known signaling target for activated Ras, PI3K (19), seems to be essential, because treatment of SKBR3 cells with the PI3K inhibitor LY294002 blocked the ability of EGF to induce the up-regulation of tTG in SKBR3 cells. This is consistent with our earlier findings that PI3K activation is necessary for tTG expression (10). Likewise, inhibiting PI3K activity prevented the combination of Ras and Cdc42 from up-regulating tTG expression (Fig. 2D).

Because our findings suggested that a signaling target capable of responding to one or more different Rho GTPases contributes to the EGF-induced expression of tTG, we also examined NFκB. We felt that NFκB represented an attractive candidate, because it is activated by Cdc42, Rac1, and RhoA and participates in the malignant transformation of breast-cancer cells (20, 21). Indeed, the results shown in Fig. 2 C and D show that blocking NFκB activation using the small molecule BAY 11-7082, which prevents the phosphorylation and ensuing degradation of the NFκB-negative regulator, IκBα, significantly compromised the ability of EGF as well as the combination of activated Ras and Cdc42 to up-regulate tTG expression.

Overexpression of tTG in SKBR3 Cells Mimics the Growth-Promoting Effects of EGF.

Because the induction of tTG expression by EGF is required for its growth-promoting effects in SKBR3 cells, we were interested in determining whether or not overexpressing tTG in these cells was sufficient to mirror the actions of this growth factor. Therefore, SKBR3 cells that stably expressed either vector alone, Myc-tagged wild-type tTG (tTG WT) or a Myc-tagged transamidation-defective form of tTG (tTG C277V), were generated. The expression of the different tTG constructs was similar in these cells (Fig. 3A Top), and as expected, the overexpression of tTG WT was accompanied by clearly detectable transamidation activity when assaying the incorporation of BPA into cell lysate proteins. However, the activity in cells expressing the tTG C277V mutant was similar to that of the vector-control cells (Fig. 3A Bottom). Immunofluorescent analysis of the stable cell lines revealed that both tTG WT and tTG C277V exhibited a similar cytoplasmic, nonnuclear distribution, indicating that eliminating the ability of tTG to transamidate substrates does not affect its cellular localization (Fig. 3B).

Fig. 3.

Overexpression of tTG in SKBR3 cells mimics the growth-promoting effects of EGF. (A) Extracts of SKBR3 cells stably expressing the vector or Myc-tagged forms of tTG WT and tTG C277V were immunoblotted with tTG and actin antibodies (Top) and assayed for transamidation activity by the incorporation of BPA into lysate proteins. An autoradiogram of these results is shown (Bottom). (B) Immunofluorescence was performed on the stable cell lines using a Myc antibody and DAPI. (C) Equal numbers of each stable cell line were seeded and maintained in medium containing 5% FBS. The cells were counted at the indicated times, and their growth rates were recorded. (D) Anchorage-independent growth assays were performed on the stable cell lines. The results from three separate assays were averaged together and graphed. Insets are images of the assays.

These stable cell lines were used to examine whether or not tTG overexpression is sufficient to mimic the actions of EGF and enhance the transformed phenotype of SKBR3 cells. Fig. 3C shows that SKBR3 cells overexpressing tTG WT had a marked growth advantage over the vector-control cells, whereas the growth rates of cells expressing the tTG C277V mutant were similar to control cells. Moreover, overexpression of tTG WT in SKBR3 cells resulted in a 2-fold increase in 5-bromo-deoxyuridine (BrdU) incorporation over control cells (Fig. S5A) as well as provided a protective effect from doxorubicin-induced apoptosis (Fig. S5B). We then examined the ability of the different stable cell lines to form colonies in soft agar. Consistent with our findings with parental SKBR3 cells (Fig. S1C), SKBR3 cells expressing the vector alone were capable of forming colonies in soft agar (Fig. 3D Inset, graph). The overexpression of tTG WT resulted in an ∼4-fold increase in colony formation, and the average size of the colonies was larger than the background colonies formed by the control cells. In contrast, SKBR3 cells expressing tTG C277V failed to grow under anchorage-independent conditions (Fig. 3D Inset, graph). In fact, the transamidation-defective tTG C277V mutant behaved like a dominant-negative inhibitor by effectively eliminating anchorage-independent growth such that it was well below the basal levels observed in the vector-control cells. Tumor-formation assays performed in mice using these same stable cell lines yielded results that further showed that tTG enhanced the transformed phenotype of SKBR3 cells (Fig. S5C).

Ability of tTG to Enhance the Growth and Transforming Phenotypes of Breast-Cancer Cells Is Caused by Its Activation of Src.

How does increasing the levels of tTG expression in breast-cancer cells enhance their oncogenic potential? To address this question, we asked whether or not overexpressing tTG in SKBR3 cells led to the activation of signaling proteins that have been implicated in cell growth and transformation. Extracts were prepared from serum-deprived SKBR3 cells stably expressing vector alone, tTG WT, or the transamidation-defective tTG C277V. They were then immunoblotted with antibodies that recognize the activated forms of different mitogenic-signaling proteins. We found that the overexpression of tTG in SKBR3 cells did not increase the activities of AKT or S6K, nor did it activate the MAP-kinases JNK, ERK, or p38 relative to their activities in vector-control cells (Fig. S6A). However, when the same cell extracts were subjected to immunoblot analysis using an anti–phospho-tyrosine antibody, a protein doublet with an apparent molecular mass in the 60–65 kDa area was enhanced in SKBR3 cells overexpressing tTG WT (Fig. S6 B and C). Because c-Src is a phospho-tyrosine–containing protein with M_r ∼60 kDa, we examined whether or not tTG might be capable of enhancing the auto-phosphorylation of Src in cells. Indeed, we found that there was a more than 2-fold increase in Src auto-phosphorylation (i.e., a measure of Src activation) in cells expressing tTG WT compared with the vector-control cells (Fig. 4A Upper), whereas in cells expressing the tTG C277V mutant, the extent of Src activation was about one-half of that for control cells. Fig. 4B shows that the magnitude and duration of EGF-dependent Src activation in SKBR3 cells was also potentiated by tTG overexpression. We then showed that blocking Src activity using the small molecule PP2 inhibited the anchorage-independent growth of SKBR3 cells caused by tTG overexpression (Fig. 4C) as well as EGF stimulation (Fig. 4D). Thus, the ability of tTG to stimulate Src kinase activity is directly responsible for the role that it plays in the EGF-stimulated growth and transformation of breast-cancer cells.

Fig. 4.

tTG-stimulated Src tyrosine kinase activity promoted aberrant cell growth. (A) Immunoprecipitations with a total-Src antibody (IP: Total Src) were performed on extracts of the indicated SKBR3 stable cell lines, and the resulting immunocomplexes were immunoblotted with phospho-Src and total-Src antibodies. The extent of Src activity detected in each sample was quantified using ImageJ software and listed below its corresponding band in the Anti-Phospho-Src blot (Fold Phospho-Src). To confirm that equal amounts of each cell extract were used for the immunoprecipitations, the whole-cell extracts were immunoblotted with Myc and actin antibodies (Input). (B) tTG stable cell lines deprived of serum were stimulated with EGF for increasing lengths of time and then lysed. The cell extracts were immunoblotted with the indicated antibodies. (C and D) SKBR3 cells overexpressing vector or tTG WT (C) or parental SKBR3 cells treated without or with EGF (D) were incubated without or with 5 μM PP2. Then, they were subjected to anchorage-independent growth assays. The results from three separate assays were averaged together and graphed.

tTG Activates Src Through Its Interaction with Keratin-19.

We set out to determine how tTG influences Src kinase activity. Although we were able to coimmunoprecipitate tTG with HA-tagged c-Src from SKBR3 cells (Fig. 5A) as well as from BT20 and MDA-MB-468 cells (Fig. S7A), we were unable to show a direct interaction between purified recombinant tTG and c-Src under conditions where we could detect an interaction between tTG and one of its best-known binding partners, fibronectin (Fig. S7B) (22). This suggested that an intermediate protein was involved in mediating the effects of tTG on Src activity. Given that only transamidation-competent tTG, and not a transamidation-defective mutant, was capable of increasing Src activity and enhancing the transforming phenotypes of breast-cancer cells, we wanted to search for candidate transamidation substrates of tTG that might serve to mediate the tTG-dependent regulation of Src. Thus, we used Myc-tagged tTG WT and the transamidation-defective tTG C277V mutant to screen lysates of SKBR3 cells for tTG-interacting proteins. The idea was that candidate substrates should be able to form a stable acyl-intermediate with the active-site cysteine residue of tTG WT but not with the C277V mutant. The ectopically expressed forms of tTG were immunoprecipitated from these extracts using an anti-Myc antibody, and then, the resulting immunocomplexes were resolved by SDS/PAGE and silver stained to detect proteins that coimmunoprecipitated with the Myc-tagged tTG constructs. Two proteins of M _r, ∼40 kDa and ∼45 kDa, were specifically associated with Myc-tagged tTG WT (Fig. 5B, arrows). The ∼45 kDa protein was determined to be actin by coimmunoprecipitation and subsequent immunoblot analysis with an anti-actin antibody, a known transamidation substrate of tTG (23). The identity of the smaller protein was initially determined by sequencing (Fig. S8A), and it was subsequently confirmed by coimmunoprecipitation and immunoblot analysis (Fig. 5C) to be the intermediate filament protein keratin-19 (K19). We also determined that K19 associates with tTG in two additional breast-cancer cell lines, namely in BT20 and MDA-MB-468 cells (Fig. S8B). Moreover, we showed that tTG is capable of labeling K19 with BPA in an in vitro transamidation assay (Fig. S9), indicating that tTG can both bind and transamidate K19.

Fig. 5.

A tTG–K19 interaction enhances the ability of tTG to bind to and stimulate Src kinase activity. (A) Immunoprecipitations with a HA antibody (IP: HA) were performed on the extracts of SKBR3 cells coexpressing HA-tagged Src and either the vector or Myc-tagged tTG WT. The resulting immunocomplexes were immunoblotted with Myc and HA antibodies. These cell extracts were also immunoblotted with Myc and HA antibodies (Input). (B and C) Immunoprecipitations with an Myc antibody (IP: Myc) were performed on extracts of SKBR3 cells stably expressing the vector, Myc-tagged tTG WT, or Myc-tagged tTG C277V. The resulting immunocomplexes were resolved by SDS/PAGE and then either (B) silver stained to detect proteins that coimmunoprecipitated with Myc-tTG WT (the proteins indicated with arrows were determined to be actin and K19) or (C) immunoblotted with K19 and Myc antibodies. To confirm that equal amounts of each cell extract were used for the immunoprecipitations, the whole-cell extracts were immunoblotted with a K19 antibody (Input). (D) HA immunoprecipitations were performed on the extracts of SKBR3 cells coexpressing Myc-tTG and HA-Src and transfected with either control siRNA or K19 siRNAs (denoted K19 siRNA-1 or K19 siRNA-2). The immunocomplexes (IP: HA) were immunoblotted with Myc and HA antibodies. These cell extracts were also immunoblotted with the indicated antibodies (Input). (E) Total Src immunoprecipitations were performed on lysates of SKBR3 cells expressing Myc-tTG WT and transfected with either control or K19 siRNAs. The immunocomplexes (IP: Total Src) were immunoblotted with phospho-Src and total-Src antibodies. The cell extracts were also immunoblotted with K19, Myc, and actin antibodies (Input). The extent of Src activity detected in each sample was quantified using ImageJ software and listed below its corresponding band in the Anti-Phospho-Src blot (Fold Phospho-Src). (F) SKBR3 cells stably expressing the vector or Myc-tTG WT were transfected with control siRNA or K19 siRNAs (Inset) and then subjected to anchorage-independent growth assays. The results from three separate assays were averaged together and graphed (graph).

Interestingly, K19 is a human breast-tumor marker (24 –26). This, coupled with the recent finding that the related family member K17 can promote epithelial cell growth by acting as a scaffold protein to bring together the necessary signaling proteins to trigger the PI3K/AKT- and mTOR-dependent activation of p70 ribosomal S6 kinase (27), led us to consider the possibility that K19 may serve to mediate the tTG-dependent activation of c-Src. This, in fact, turned out to be the case, as shown by the data in Fig. 5D, where the ability of tTG to be coimmunoprecipitated with HA-tagged Src from SKBR3 cells was dependent on K19 (i.e., knock-downs of K19 blocked the ability of tTG to interact with Src). Moreover, the ability of tTG to activate Src is dependent on K19 as indicated by the loss of this activation when K19 expression is knocked-down by RNAi (Fig. 5E Upper). Additionally, K19 is necessary for the ability of tTG to enhance the anchorage-independent growth of SKBR3 cells (Fig. 5F).

Discussion

Excessive activation of the EGFR contributes to oncogenesis by stimulating the growth and survival of cancer cells, whereas small molecule inhibitors or antibody-based approaches that target the EGFR or its immediate downstream effectors have shown some promise as treatments for certain human cancers (1, 2, 4, 5). Therefore, continuing efforts to better understand the mechanisms by which the EGFR stimulates cancer progression will play a critical part in the development of alternative strategies to treat cancers. Although it is generally appreciated that the EGFR modifies global gene-expression patterns in cells, the identities of those gene products whose induction is most important for cell growth and/or survival are largely unknown. Here, we have identified tTG, a dual-function GTPase/acyl transferase, as one such protein. The initial indication for this came from the finding that stimulating SKBR3 or BT20 breast-cancer cells with EGF not only increased their proliferative capacity and ability to form colonies in soft agar, but it also resulted in the induction of tTG expression and activation. Suppressing tTG expression or activity potently inhibited the EGF-induced growth advantages in each of these cell lines. We further showed that overexpression of tTG WT in SKBR3 cells could fully recapitulate the growth-stimulatory actions of EGF, which implicates tTG as a key participate in EGFR-promoted cellular transformation.

Thus far, very little has been known about the signaling pathways that mediate EGF-induced tTG expression. However, we have begun to delineate some of the events that must occur to stimulate tTG expression and cellular transformation, which are summarized in Fig. 6. Although the individual ectopic expression of activated forms of various signaling proteins in SKBR3 cells did not induce tTG expression, we found that the Ras- and Cdc42-signaling pathways cooperated to up-regulate tTG. We then showed that the induction of tTG expression by either EGF stimulation or coexpression of activated Ras and Cdc42 was sensitive to the PI3K inhibitor LY294002 as well as the NFκB inhibitor BAY 11-7082.

Fig. 6.

Diagram depicting how tTG participates in an EGFR/Src signaling pathway that leads to enhanced cancer-cell growth.

Increasing the levels of tTG expression in SKBR3 breast-cancer cells enhances their oncogenic potential, and thus, it mirrors the actions of EGF treatment. Surprisingly, we discovered that this is caused by the ability of tTG to activate c-Src. Consistent with these results, as well as with several lines of evidence linking Src kinase activity to tumor progression, the anchorage-independent growth advantage afforded to SKBR3 cells by either EGF stimulation or tTG overexpression was severely compromised when these cells were cultured in the presence of the Src inhibitor PP2. Overall, these findings highlight an interesting connection between the overexpression of tTG observed in increasing numbers of breast cancer as well as other human cancers and the presence of elevated Src activity.

An intriguing question concerns how tTG expression leads to increased Src activity in cells. We obtained a clue when we found that Src coimmunoprecipitated with tTG from SKBR3 cell lysates but that the recombinant forms of these two proteins failed to show a similar association in vitro. This implied that the tTG–Src interaction in cells was indirect and most likely mediated by another protein. Indeed, we identified the intermediate filament K19 as a tTG-binding partner that is essential for tTG’s ability to associate with Src in cells as well as the enhanced Src activity observed in the SKBR3 cells stably overexpressing tTG. Moreover, we showed that K19 is capable of being transamidated by tTG in vitro. Thus, the tTG-catalyzed cross-linking of K19 might enable it to act as a signaling scaffold that binds and favors the activated state of c-Src, an idea that we are currently investigating. Interestingly, K19 belongs to a family of cytokeratins, many of which, including K19, are overexpressed in a large number of human cancer types (28, 29) that are routinely used as diagnostic indicators of metastasis and tumor aggressiveness (24 –26). Given the connections between K19 and human cancers, an important question has been whether or not the cytokeratins directly contribute to oncogenesis. Our findings that K19 serves to bridge the interaction between tTG and Src (Fig. 6), resulting in enhanced Src kinase activity and aberrant cell growth, point to a rather unexpected role for K19 in EGFR-promoted cellular transformation.

In summary, the findings presented in this study show that the induction of tTG expression and activation by EGF are both necessary and sufficient for mediating the growth-promoting actions of EGF in human breast-cancer cells. Interestingly, a recent proteomic screen of the U87 brain-tumor cell line overexpressing a highly oncogenic form of the EGFR known as EGFR variant type III (EGFRvIII) showed that tTG was one of three proteins whose expression was enhanced by this mutant EGFR (30). Whether or not tTG is important for promoting the oncogenic potential of EGFRvIII remains to be determined. However, our findings now point to an important relationship between EGFR-mediated transformation and tTG expression, and they suggest that tTG may represent a potentially valuable therapeutic target.

Materials and Methods

Materials.

Cell-culture reagents, EGF, Lipofectamine, Lipofectamine 2000, protein G beads, glutathione-agarose beads, tTG and K19 RNAis, and the HA, GST, and Myc antibodies were from Invitrogen. LY 294002, PP2, PD 98059, BAY 11-7082, DAPI, and MDC were from Calbiochem. The 5-(biotinamido) pentylamine was from Pierce. The tTG, actin, and K19 antibodies were from Neomarkers, whereas antibodies recognizing total or activated Src, AKT, JNK, P38, p70 S6-kinase, and ERK as well as the anti–phospho-tyrosine antibody were from Cell Signaling.

Cell Culture.

SKBR3 and BT20 cell lines were grown in RPMI medium 1640 containing 10% FBS. Expression constructs were transfected into cells using Lipofectamine, whereas RNAis were introduced into cells using Lipofectamine 2000. Cells stably expressing Myc-tagged pcDNA3 vector, Myc-tTG WT, or Myc-tTG C277V were generated by selection with growth medium supplemented with 800 μg/mL G418. Where indicated, cells were treated with various combinations of 0.1 μg/mL EGF, 20 μM MDC, 10 μM LY294002, 20 μM PD98059, 2 μM BAY 11-7082, or 5 μM PP2.

Immunoblot Analysis.

Cells were lysed with lysis buffer (25 mM Tris, 100 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM DTT, 1 mM NaVO₄, 1 mM β-glycerol phosphate, and 1 μg/mL aprotinin). Then, the lysates were resolved by SDS/PAGE, and the proteins were transferred to polyvinylidene fluoride membranes. The filters were incubated with the various primary antibodies diluted in 20 mM Tris, 135 mM NaCl, and 0.02% Tween 20. The primary antibodies were detected with horseradish peroxidase–conjugated secondary antibodies followed by exposure to ECL reagent.

Immunofluorescence.

SKBR3 cells expressing the Myc-pCDNA3 vector, Myc-tTG WT, or Myc-tTG C277V were fixed using 5% formaldehyde, permeabilized with 0.2% Triton X-100, and then incubated with a Myc antibody. The Myc antibody was detected using an Oregon green 488-conjugated secondary antibody, and DAPI was used to stain nuclei.

Transamidation Assay.

These assays were performed as previously described (10).

Cell-Growth Assays.

Parental SKBR3 cells or the SKBR3 stable cell lines were plated in dishes at a density of 5 × 10⁴ cells/dish and cultured in medium containing 2% FBS without or with EGF, MDC, or PP2. Every other day for 1 week, one set of cells was collected and counted, whereas the medium on the remaining sets of cells was replenished.

Soft-Agar Assays.

Parental SKBR3 and BT20 cells or the SKBR3 stable cell lines were plated at a density of 6 × 10³ cells/mL in medium containing 0.3% agarose without or with EGF, MDC, or PP2 onto underlays composed of growth medium containing 0.6% agarose in six-well dishes. The cultures were fed 1 week later, and after 14 days of growth, the colonies were counted.

Immunoprecipitation.

Cell extracts (typically 600 μg) were precleared with protein G beads before incubating with a particular antibody for 2 h, followed by the addition of protein G beads for 1 h. The beads were washed with cell-lysis buffer before being boiled with Laemmli’s sample buffer and subjected to SDS/PAGE and Western blotting.

Silver Staining and MS-Peptide Sequence Identification.

Silver staining of proteins resolved by SDS/PAGE was performed based on the manufacturer’s instructions (Bio-Rad). Protein bands of interest were excised from the gel and washed three times with 50% acetonitrile. The samples were analyzed at the Harvard Microchemistry and Proteomics Analysis Facility by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry on a Thermo LTQ–Orbitrap mass spectrometer.