Bruce E. Elliott

The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells

IntroductionThe membrane cytoskeletal crosslinker ezrin participates in several functions including cell adhesion, motility and cell survival, and there is increasing evidence that it regulates tumour progression. However, the role played by ezrin in breast cancer metastasis has not been clearly delineated.MethodsWe examined the role of ezrin in metastasis using a highly metastatic murine mammary carcinoma cell line, namely AC2M2. Stable cell clones that overexpress wild-type ezrin or a dominant-negative amino-terminal domain of ezrin were selected. They were then tested for cell motility and invasion in vitro, and metastasis in a mouse in vivo tumour transplantation model.ResultsParental AC2M2 cells and cells overexpressing wild-type ezrin were transplanted into the mammary fat pad of syngeneic recipient mice; these animals subsequently developed lung metastases. In contrast, expression of the dominant-negative amino-terminal ezrin domain markedly inhibited lung metastasis. Consistent with this effect, we observed that the expression of amino-terminal ezrin caused strong membrane localization of cadherin, with increased cell–cell contact and a decrease in cell motility and invasion, whereas cells expressing wild-type ezrin exhibited strong cytoplasmic expression of cadherins and pseudopodia extensions. In addition, inhibitors of phosphatidylinositol 3-kinase and c-Src significantly blocked cell motility and invasion of AC2M2 cells expressing wild-type ezrin. We further found that overexpression of amino-terminal ezrin reduced levels of Akt pS473 and cytoskeletal-associated c-Src pY418 in AC2M2 cells, which contrasts with the high levels of phosphorylation of these proteins in cells expressing wild-type ezrin. Phosphorylated Erk1/2 was also reduced in amino-terminal ezrin expressing cells, although a mitogen-activated protein kinase kinase (MEK) inhibitor had no detectable effect on cell motility or invasion in this system.ConclusionOur findings indicate that ezrin is required for breast cancer metastasis, and that c-Src and phosphatidylinositol 3-kinase/Akt are effectors of ezrin in the cell motility and invasion stages of the metastatic process. Together, these results suggest that blocking ezrin function may represent a novel and effective strategy for preventing breast cancer metastasis.

Osteopontin‐induced, integrin‐dependent migration of human mammary epithelial cells involves activation of the hepatocyte growth factor receptor (Met)

Osteopontin (OPN) is a secreted glycophosphoprotein which induces migration of mammary carcinoma cells, and has been implicated in the malignancy of breast carcinoma. Hepatocyte growth factor (HGF) induces cell migration of several mammary epithelial cell (MEC) lines, via activation of its cognate receptor (Met). This study examines the mechanism of OPN‐induced MEC migration, in terms of the cell surface integrins involved and induction of the HGF/Met pathway. Three different MEC cell lines were used, representing different stages of tumor progression: 21PT, non‐tumorigenic; 21NT, tumorigenic; non‐metastatic; and MDA‐MB‐435, tumorigenic, highly metastatic. Human recombinant OPN was found to induce the migration of all three lines. OPN‐induced migration of 21PT and 21NT cells was αvβ5 and β1‐integrin dependent, and αvβ3‐independent, while that of MDA‐MB‐435 cells was αvβ3‐dependent. HGF also induced migration of all three cell lines, and a synergistic response was seen to HGF and OPN together. The increased migration response to OPN was found to be associated with an initial increase in Met kinase activity (within 30 min), followed by an increase in Met mRNA and protein expression. OPN‐induced cell migration is thus mediated by different cell surface integrins in MEC lines representing different stages of progression, and involves activation of the HGF receptor, Met. J. Cell. Biochem. 78:465–475, 2000.

Perspectives on the Role of Mhc Antigens in Normal and Malignant Cell Development

Publisher Summary This chapter discusses the basic properties of major histocompatibility complex (MHC) biology, function, and the possible mechanisms of MHC regulation in normal and malignant cells. The influence of tissue microenvironment and of tissue-specific lymphocyte migration on host–tumor immune interactions is also discussed. Class I MHC antigens are expressed on nearly all adult tissues, though at varying levels. The organ with the highest MHC antigen expression is the spleen, followed by other lymphoid organs. Transplantation of low class I MHC-expressing tumor cell lines into syngeneic or nude mouse recipients can result in augmentation of tumor-derived class I antigens. The in situ levels of class I MHC can be monitored in experiments designed to examine the role of MHC in malignancy. The chapter examines several studies of MHC expression in metastasis. One of them demonstrates an imbalance of D- versus K-antigen expression in metastatic lines derived from the T10 sarcoma and the 3LL carcinoma, respectively.

Cell-to-cell adhesion modulates Stat3 activity in normal and breast carcinoma cells.

Stat3 (signal transducer and activator of transcription-3) activity is required for transformation by a number of oncogenes, while a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. Although in most instances Stat3 is growth-promoting, the impact of cell density on Stat3 activation status and the biological importance of Stat3 during growth arrest have not been characterized. Previous results indicated that cell density alters tyrosine phosphorylation levels of cultured cells. Since signalling through Stat3 is determined by a key phosphorylation at tyr705, we examined the effects of cell density upon Stat3 activity in normal breast epithelial cells, breast carcinoma lines and normal mouse fibroblasts. Intriguingly, the results revealed a dramatic increase in Stat3, tyr705 phosphorylation and activity with cell density, which gradually declined at later stages. This activation was dependent upon cell–cell contact, since it was eliminated if cell adhesion was disrupted through calcium chelation, while it was reinstated through cell aggregation. Furthermore, this activation was suppressed following inhibition of JAKs (Janus kinases) but not inhibition of Fer, IGF1-R, or kinases of the c-Src family. On the other hand, constitutively active Stat3 in carcinoma lines, known to harbor activated Src, was blocked by pharmacological inhibitors of Src as well as JAKs. These results point to the existence of two distinct pathways of Stat3 activation in breast carcinomas, based on Src dependence. More importantly, our results suggest that Stat3 activity is upregulated during the confluence-mediated growth arrest by a signalling mechanism that requires JAKs.

Co-operative Effect of c-Src Tyrosine Kinase and Stat3 in Activation of Hepatocyte Growth Factor Expression in Mammary Carcinoma Cells

We have previously shown coexpression of hepatocyte growth factor (HGF) and its receptor Met in the invasive tumor front of human breast carcinomas. We have also demonstrated secretion of HGF, constitutive activation of Met, and increased invasion in a murine breast carcinoma cell line, SP1. These observations suggest the presence of an HGF autocrine loop in some breast carcinoma cells, which confers increased survival, growth, and invasiveness during tumor progression and metastasis. c-Src tyrosine kinase, which is critical in regulating the expression of many genes, is activated in SP1 carcinoma cells, as well as in most human breast cancers. We therefore examined the role of c-Src kinase in HGF expression in breast carcinoma cells. Expression of activated c-Src in SP1 cells increased transcription from the HGF promoter and expression of HGF mRNA and protein, while dominant negative c-Src had the opposite effect. Using deletion analysis, we showed that the region between −254 and −70 base pairs was required for c-Src responsiveness of the HGF promoter. This region contains two putative consensus sequences (at −110 and −149 base pairs) for the Stat3 transcription factor, which bind protein complexes containing Stat3 (but not Stat1, -5A, or -5B). Coexpression of activated c-Src and Stat3 synergistically induced strong HGF promoter activity in SP1 cells, as well as in a nonmalignant epithelial cell line, HC11 (HGF negative). c-Src kinase activity correspondingly increased the tyrosine 705 phosphorylation and DNA binding affinity of Stat3 (but not Stat1, -5A, or -5B). Collectively, our data indicate a cooperative effect of c-Src kinase and Stat3 in the activation of HGFtranscription and protein expression in breast carcinoma cells. This process may be important in overriding the strong repression ofHGF expression in nonmalignant epithelium, and thereby promote tumorigenesis.

A Novel RET Kinase–β-Catenin Signaling Pathway Contributes to Tumorigenesis in Thyroid Carcinoma

The RET receptor tyrosine kinase has essential roles in cell survival, differentiation, and proliferation. Oncogenic activation of RET causes the cancer syndrome multiple endocrine neoplasia type 2 (MEN 2) and is a frequent event in sporadic thyroid carcinomas. However, the molecular mechanisms underlying RETs potent transforming and mitogenic signals are still not clear. Here, we show that nuclear localization of beta-catenin is frequent in both thyroid tumors and their metastases from MEN 2 patients, suggesting a novel mechanism of RET-mediated function through the beta-catenin signaling pathway. We show that RET binds to, and tyrosine phosphorylates, beta-catenin and show that the interaction between RET and beta-catenin can be direct and independent of cytoplasmic kinases, such as SRC. As a result of RET-mediated tyrosine phosphorylation, beta-catenin escapes cytosolic down-regulation by the adenomatous polyposis coli/Axin/glycogen synthase kinase-3 complex and accumulates in the nucleus, where it can stimulate beta-catenin-specific transcriptional programs in a RET-dependent fashion. We show that down-regulation of beta-catenin activity decreases RET-mediated cell proliferation, colony formation, and tumor growth in nude mice. Together, our data show that a beta-catenin-RET kinase pathway is a critical contributor to the development and metastasis of human thyroid carcinoma.

Phosphatidylinositol 3-kinase activity is required for hepatocyte growth factor-induced mitogenic signals in epithelial cells.

Phosphatidylinositol (PI) 3-kinase is an important enzyme implicated in growth factor-stimulated intracellular signaling. In this study we have shown that hepatocyte growth factor (HGF) induces a rapid tyrosine phosphorylation of PI 3-kinase and association with HGF receptor/Met in Mv1Lu epithelial cells. Murine mammary carcinoma (SP1) cells, which co-express HGF and HGF receptor/Met, showed sustained phosphorylation of PI 3-kinase. Wortmannin, a potent inhibitor of PI 3-kinase, inhibited HGF-induced PI 3-kinase activity, proliferation of Mv1Lu cells, and spontaneous growth of SP1 cells in a dose-, and time-dependent manner. Transfection of a dominant negative mutant p85 (Δp85) subunit of PI 3-kinase into SP1 cells strongly inhibited HGF-stimulated proliferation and PI 3-kinase activity. However, wortmannin did not influence HGF-induced c-Jun expression. Furthermore, HGF stimulated S6 kinase activity, but its activity was not required for HGF-induced proliferation. Overall, these results suggest that HGF-induced PI 3-kinase activity is important for the mitogenic action of HGF in epithelial cells and further demonstrate that expression of c-Jun is not influenced by inhibition of PI 3-kinase activity.

Autocrine secretion of TGF-β1 and TGF-β2 by pre-adipocytes and adipocytes: A potent negative regulator of adipocyte differentiation and proliferation of mammary carcinoma cells

SummaryWe have developed an in vitro system to examine the influence of adipocytes, a major mammary stromal cell type, on the growth of a murine mammary carcinoma, SP1. Previously, we have shown that 3T3-L1 adipocytes release a mitogenic factor, hepatocyte growth factor, which strongly stimulates proliferation of SP1 cells. We now show that 3T3-L1 pre-adipocytes secrete active inhibitory molecules which inhibit DNA synthesis in SP1 cells. In addition, latent inhibitory activity is present in conditioned media (CM) from both pre-adipocytes and adipocytes, and is activated following acid treatment. CM also inhibited DNA synthesis in Mv1Lu wild type epithelial cells, but not DR27 mutant epithelial cells which lack TGF-β type II receptor. Inhibitory activity of CMs was partially abrogated by neutralizing anti-TGF-β1 and anti-TGF-β2 antibodies, and was removed following ultrafiltration through membranes of 10 000 Mr but not 30 000 Mr pore size. These results show that the inhibitory effect on DNA synthesis is mediated by TGF-β1-like and TGF-β2-like molecules. In addition, acid-treated CM as well as purified TGF-β inhibited differentiation of pre-adipocytes. Untreated pre-adipocyte CM, but not mature adipocyte CM, spontaneously inhibited adipocyte differentiation. Together, these findings indicate that pre-adipocytes spontaneously activate their own secreted TGF-β, whereas mature adipocytes do not, and suggest that activation of TGF-β has a potent negative regulatory effect on adipocyte differentiation and tumor growth. Thus, TGF-β may be an important modulator of tumor growth and adipocyte differentiation via both paracrine and autocrine mechanisms. These findings emphasize the importance of adipocyte-tumor interactions in the regulation of tumor microenvironment.

An enzyme-linked immunosorbent assay (Elisa) for detergent solubilized Ia glycoproteins using nitrocellulose membrane discs

An enzyme-linked immunosorbent assay has been developed for the quantitative detection of detergent solubilized murine Ia. Nitrocellulose membrane discs were used to bind membrane glycoproteins applied in solutions containing detergent. The bound antigen was detected by monoclonal antibodies and horseradish-peroxidase-coupled anti-IgG. The assay produced a linear response with respect to antigen concentration, and could readily detect partially purified Ia derived from 10(3) to 10(4) mitogen stimulated spleen cells. Nitrocellulose discs efficiently bound protein in the presence of deoxycholate, taurocholate, and octylglucoside. Less binding occurred in the presence of Triton X-100 or Tween 80, but 90% binding efficiency was obtained in 0.01% solutions of these detergents. The association of protein with the discs was stable under normal conditions for antigen detection, but could be further stabilized by briefly fixing with glutaraldehyde for more rigorous procedures. The ability of this method to detect antigen in detergent solutions makes it useful in monitoring fractions during the purification of cell membrane proteins.

Co-operative effect of c-Src and ezrin in deregulation of cell-cell contacts and scattering of mammary carcinoma cells.

The non‐receptor tyrosine kinase c‐Src is activated in many human cancer types, and induces deregulation of cadherin‐based cell–cell contacts and actin cytoskeleton. Because ezrin, a protein which cross‐links the plasma membrane with the actin cytoskeleton, is often over‐expressed in human cancers, and participates in cell adhesion, motility, and cell scattering, we therefore investigated whether c‐Src co‐operates with ezrin in regulating cell–cell contacts in a murine mammary carcinoma cell line, SP1. SP1 cells over‐expressing wild type ezrin, or an activated c‐Src mutant, formed loose aggregates which scattered spontaneously when plated on plastic. When wild type ezrin and activated c‐Src were co‐expressed, scattering was increased, cell–cell contacts disrupted, and cell aggregation prevented. Pre‐treatment with the c‐Src family kinase inhibitor PP2 partially restored aggregation of cells expressing activated c‐Src and wild type ezrin, indicating that c‐Src family kinases act co‐operatively with ezrin in regulating cell–cell contacts. Furthermore, expression of a truncated NH2‐terminal domain of ezrin, which has dominant negative function, blocked the cell scattering effect of activated c‐Src and promoted formation of cohesive cell–cell contacts. Together, these results suggest co‐operativity between c‐Src and ezrin in deregulation of cell–cell contacts and enhancing scattering of mammary carcinoma cells.