Defining MAP3 kinases required for MDA-MB-231 cell tumor growth and metastasis.

Analysis of patient tumors suggests that multiple MAP3 kinases (MAP3Ks) are critical for growth and metastasis of cancer cells. MAP3Ks selectively control the activation of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal kinase (JNK), p38 and ERK5 in response to receptor tyrosine kinases and GTPases. We used MDA-MB-231 cells because of their ability to metastasize from the breast fat pad to distant lymph nodes for an orthotopic xenograft model to screen the function of seven MAP3Ks in controlling tumor growth and metastasis. Stable short hairpin RNA (shRNA) knockdown was used to inhibit the expression of each of the seven MAP3Ks, which were selected for their differential regulation of the MAPK network. The screen identified two MAP3Ks, MEKK2 and MLK3, whose shRNA knockdown caused significant inhibition of both tumor growth and metastasis. Neither MEKK2 nor MLK3 have been previously shown to regulate tumor growth and metastasis in vivo. These results demonstrated that MAP3Ks, which differentially activate JNK, p38 and ERK5, are necessary for xenograft tumor growth and metastasis of MDA-MB-231 tumors. The requirement for MAP3Ks signaling through multiple MAPK pathways explains why several members of the MAPK network are activated in cancer. MEKK2 was required for epidermal growth factor receptor and Her2/Neu activation of ERK5, with ERK5 being required for metastasis. Loss of MLK3 expression increased mitotic infidelity and apoptosis in vitro. Knockdown of MEKK2 and MLK3 resulted in increased apoptosis in orthotopic xenografts relative to control tumors in mice, inhibiting both tumor growth and metastasis; MEKK2 and MLK3 represent untargeted kinases in tumor biology for potential therapeutic development.

Persistent transactivation of EGFR and ErbB2/HER2 by protease-activated receptor-1 promotes breast carcinoma cell invasion.

Hyperactivation of ErbB signaling is implicated in metastatic breast cancer. However, the mechanisms that cause dysregulated ErbB signaling and promote breast carcinoma cell invasion remain poorly understood. One pathway leading to ErbB activation that remains unexplored in breast carcinoma cell invasion involves transactivation by G-protein-coupled receptors (GPCRs). Protease-activated receptor-1 (PAR1), a GPCR activated by extracellular proteases, is overexpressed in invasive breast cancer. PAR1 is also proposed to function in breast cancer invasion and metastasis, but how PAR1 contributes to these processes is not known. In this study, we report that proteolytic activation of PAR1 by thrombin induces persistent transactivation of EGFR and ErbB2/HER2 in invasive breast carcinoma, but not in normal mammary epithelial cells. PAR1-stimulated EGFR and ErbB2 transactivation leads to prolonged extracellular signal-regulated kinase-1 and -2 signaling and promotes breast carcinoma cell invasion. We also show that PAR1 signaling through Galpha(i/o) and metalloprotease activity is critical for ErbB transactivation and cellular invasion. Finally, we demonstrate that PAR1 expression in invasive breast carcinoma is essential for tumor growth in vivo assessed by mammary fat pad xenografts. These studies reveal a critical role for PAR1, a receptor activated by tumor-generated proteases, in hyperactivation of ErbB signaling that promotes breast carcinoma cell invasion.

Role of mitogen-activated protein kinase kinase kinases in signal integration.

Mitogen-activated protein kinases (MAPKs) are members of a dynamic protein kinase network through which diverse stimuli regulate the spatio-temporal activities of complex biological systems. MAPKs regulate critical cellular functions required for homeostasis such as the expression of cytokines and proteases, cell cycle progression, cell adherence, motility and metabolism. MAPKs therefore influence cell proliferation, differentiation, survival, apoptosis and development. In vertebrates, five MAPK families are regulated by MAPK kinase kinase-MAPK kinase-MAPK (MKKK-MKK-MAPK) phosphorelay systems. There are at least 20 MKKKs that selectively phosphorylate and activate different combinations of the seven MKKs, resulting in a specific activation profile of members within the five MAPK families. MKKKs are differentially activated by upstream stimuli including cytokines, antigens, toxins and stress insults providing a mechanism to integrate the activation of different MAPKs with the cellular response to each stimulus. Thus, MKKKs can be considered as 'signaling hubs' that regulate the specificity of MAPK activation. In this review, we describe how the MKKK 'hub' function regulates the specificity of MAPK activation, highlighting MKKKs as targets for therapeutic intervention in cancer and other diseases.

Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors.

The controlled and specific re-activation of endogenous tumor suppressors in cancer cells represents an important therapeutic strategy to block tumor growth and subsequent progression. Other than ectopic delivery of tumor suppressor-encoded cDNA, there are no therapeutic tools able to specifically re-activate tumor suppressor genes that are silenced in tumor cells. Herein, we describe a novel approach to specifically regulate dormant tumor suppressors in aggressive cancer cells. We have targeted the Mammary Serine Protease Inhibitor (maspin) (SERPINB5) tumor suppressor, which is silenced by transcriptional and aberrant promoter methylation in aggressive epithelial tumors. Maspin is a multifaceted protein, regulating tumor cell homeostasis through inhibition of cell growth, motility and invasion. We have constructed artificial transcription factors (ATFs) made of six zinc-finger (ZF) domains targeted against 18-base pair (bp) unique sequences in the maspin promoter. The ZFs were linked to the activator domain VP64 and delivered in breast tumor cells. We found that the designed ATFs specifically interact with their cognate targets in vitro with high affinity and selectivity. One ATF was able to re-activate maspin in cell lines that comprise a maspin promoter silenced by epigenetic mechanisms. Consistently, we found that this ATF was a powerful inducer of apoptosis and was able to knock down tumor cell invasion in vitro. Moreover, this ATF was able to suppress MDA-MB-231 growth in a xenograft breast cancer model in nude mice. Our work suggests that ATFs could be used in cancer therapeutics as novel molecular switches to re-activate dormant tumor suppressors.

MEKK1 controls matrix degradation and tumor cell dissemination during metastasis of polyoma middle-T driven mammary cancer.

Mammary tumor cells are required to degrade the surrounding matrix and disseminate in order to metastasize, and both of these processes are controlled by a tumor cell-signaling network that remains poorly defined. MEKK1 is a MAPKKK that regulates both the extracellular signal regulated kinase (ERK1/2) and the c-Jun amino terminal kinase (JNK) signaling pathways. MEKK1 signaling regulates migration through control of cell adhesion and is required for inducible expression of urokinase-type plasminogen activator (uPA). MEKK1-deficient mice with mammary gland-targeted expression of the polyoma middle T antigen (PyMT) transgene develop primary mammary tumors at a rate and frequency similar to wild-type littermates, indicating that MEKK1 deficiency does not affect PyMT-mediated transformation. However, MEKK1-/- mice display significantly delayed tumor cell dissemination and lung metastasis. Delayed MEKK1-dependent tumor dissemination is associated with markedly reduced tumor uPA expression, gelatinase activity, and prolonged tumor basement membrane integrity. siRNA-mediated MEKK1 knockdown inhibits uPA activity, cell migration and invasion in MDA-MB-231 human breast cancer cells. Thus MEKK1 controls tumor progression by regulating both the migration and proteolysis aspects of tumor cell invasiveness. To our knowledge, this is the first example of a MAPKKK that regulates metastasis through control of tumor invasiveness.

Tyrosine phosphorylation of p85 relieves its inhibitory activity on phosphatidylinositol 3-kinase.

Under resting conditions, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) serves to both stabilize and inactivate the p110 catalytic subunit. The inhibitory activity of p85 is relieved by occupancy of the NH(2)-terminal SH2 domain of p85 by phosphorylated tyrosine. Src family kinases phosphorylate tyrosine 688 in p85, a process that we have shown to be reversed by the activity of the p85-associated SH2 domain-containing phosphatase SHP1. We demonstrate that phosphorylation of the downstream PI3K target Akt is increased in cells lacking SHP1, implicating phosphorylation of p85 in the regulation of PI3K activity. Furthermore, the in vitro specific activity of PI3K associated with tyrosine- phosphorylated p85 is higher than that associated with nonphosphorylated p85. Expression of wild-type p85 inhibits PI3K enzyme activity as indicated by PI3K- dependent Akt phosphorylation. The inhibitory activity of p85 is accentuated by mutation of tyrosine 688 to alanine and reversed by mutation of tyrosine 688 to aspartic acid, changes that block and mimic tyrosine phosphorylation, respectively Strikingly, mutation of tyrosine 688 to aspartic acid completely reverses the inhibitory activity of p85 on cell viability and activation of the downstream targets Akt and NFkappaB, indicative of the physiological relevance of p85 phosphorylation. Tyrosine phosphorylation of Tyr(688) or mutation of tyrosine 688 to aspartic acid is sufficient to allow binding to the NH(2)-terminal SH2 domain of p85. Thus an intramolecular interaction between phosphorylated Tyr(688) and the NH(2)-terminal SH2 domain of p85 can relieve the inhibitory activity of p85 on p110. Taken together, the data indicate that phosphorylation of Tyr(688) in p85 leads to a novel mechanism of PI3K regulation.

Functional evidence for an ovarian cancer tumor suppressor gene on chromosome 22 by microcell-mediated chromosome transfer.

The identity of many tumor suppressor genes important in epithelial ovarian cancer tumorigenesis remains unknown. In an effort to localize a novel tumor suppressor on chromosome 22, a psv2neo tagged human chromosome 22 was transferred into the malignant epithelial ovarian cancer cell line, SKOv-3, by microcell-mediated chromosome transfer. Complete suppression of the transformed phenotype was observed in 16 of 18 individual microcell hybrid clones as evidenced by the complete abrogation of cell growth under anchorage-independent conditions. In vitro doubling times were also dramatically reduced, as was the ability to form subcutaneous tumors in CD1 nu/nu mice. Only one polymorphic marker, D22S429, segregated with decreased transformation and tumorigenic potential, suggesting that an unrecognized tumor suppressor may localize to chromosome 22q11-q12. These data provide functional support for the presence of a novel tumor suppressor locus (or loci) on chromosome 22 that is important in ovarian cancer tumorigenesis.