Stress signals activate the SAPK/JNK and p38 MAPK classes of protein kinases, which mediate cellular responses, including steps in apoptosis and the maturation of some cell types. We now show that stress signals initiated by transforming growth factor-1 (TGF-1) induce G 1 arrest through protein stabilization of the CDK inhibitor p21Cip1 . TGF-1 was previously shown to increase p21 protein levels, which in turn mediated G 1 arrest through inactivation of the CDK2-cyclin E complex in HD3 cells (Yan, Z., Kim, G.-Y., Deng, X., and Friedman, E. (2002) J. Biol. Chem. 277, 9870 -9879). We now demonstrate that the increase in p21 abundance is caused by a post-transcriptional, SMAD-independent mechanism. TGF-1 activated p38␣ and JNK1, which initiated the phosphorylation of p21. TGF-1 treatment increased the half-life of p21 by 3-4-fold. The increase in p21 stability was detected following activation of p38␣ and JNK1, and treatment of cells with the p38 inhibitor SB203580 prevented this increase in p21 stability. p38␣ and JNK1 phosphorylated p21 in vivo, and both p38␣ and JNK1 phosphorylated p21 at Ser 130 in vitro. Peptide mapping demonstrated that both TGF-1 and p38␣ induced phosphorylation of p21 at Ser 130 in vivo, and mutation of Ser 130 to alanine rendered p21 less stable than wild-type p21. TGF-1 increased the stability of wildtype p21, but not the p21-S130A mutant. These findings demonstrate that SAPKs can mediate cell cycle arrest through post-translational modification of p21.
Breast cancer metastasis involves lymphatic dissemination in addition to hematogenous spreading. Although stromal lymphatic vessels (LVs) serve as initial metastatic routes, roles of organ-residing LVs are under-investigated. Here we show that lymphatic endothelial cells (LECs), a component of LVs within pre-metastatic niches, are conditioned by triple-negative breast cancer (TNBC) cells to accelerate metastasis. LECs within the lungs and lymph nodes, conditioned by tumor-secreted factors express CCL5 that is not expressed either in normal LECs or cancer cells, and direct tumor dissemination into these tissues. Moreover, tumor-conditioned LECs promote angiogenesis in these organs, allowing tumor extravasation and colonization. Mechanistically, tumor cell-secreted IL6 causes Stat3 phosphorylation in LECs. This pStat3 induces HIF-1α and VEGF, and a pStat3-pc-Jun-pATF-2 ternary complex induces CCL5 expression in LECs. This study demonstrates anti-metastatic activities of multiple repurposed drugs, blocking a self-reinforcing paracrine loop between breast cancer cells and LECs.
Multiple factors including long-term treatment with tamoxifen are involved in the development of selective estrogen receptor (ER) modulator resistance in ERα-positive breast cancer. Many underlying molecular events that confer resistance are known but a unifying theme is yet to be revealed. In this report, we provide evidence that HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen via cross-talk between receptor tyrosine kinases and ERα signaling. HOXB7 is an ERα-responsive gene. Extended treatment of MCF-7 cells with tamoxifen resulted in progressively increasing levels of HOXB7 expression, along with EGFR and EGFR ligands. Up-regulation of EGFR occurs through direct binding of HOXB7 to the EGFR promoter, enhancing transcriptional activity. Finally, higher expression levels of HOXB7 in the tumor significantly correlated with poorer disease-free survival in ERα-positive patients with breast cancer on adjuvant tamoxifen monotherapy. These studies suggest that HOXB7 acts as a key regulator, orchestrating a major group of target molecules in the oncogenic hierarchy. Functional antagonism of HOXB7 could circumvent tamoxifen resistance.
Mirk/Dyrk1B is an arginine-directed serine/threonine protein kinase that is expressed at low levels in most normal tissues but at elevated levels in many tumor cell lines and in normal skeletal muscle. Colon carcinoma cell lines stably overexpressing Mirk proliferated in serumfree medium, but the mechanism of Mirk action is unknown. DCoHm (dimerization cofactor of hepatocyte nuclear factor 1␣ ( HNF1␣) from muscle), a novel gene of the DCoH family with 78% amino acid identity to DCoH, was identified as a Mirk-binding protein by yeast two-hybrid analysis and cloned. Mirk co-immunoprecipitated with DCoHm and bound to DCoHm in glutathione S-transferase pull-down assays. DCoH stabilizes HNF1␣ as a dimer and enhances its transcriptional activity on the -fibrinogen promoter reporter, and DCoHm had similar activity. Mirk enhanced HNF1␣ transcriptional activity in a dosedependent manner, whereas two kinase-inactive Mirk mutants and a Mirk N-terminal deletion mutant did not. Mirk, DCoHm, and HNF1␣ formed a complex. Mirk bound to a specific region within the CREB-binding proteinbinding region of HNF1␣ and phosphorylated HNF1␣ at a site adjacent to the Mirk-binding region. Conversely, the HNF1␣ binding domain was located within the first five conserved kinase subdomains of Mirk. Mirk co-immunoprecipitated with the MAPK kinase MKK3, an upstream activator of p38. MKK3 enhanced Mirk kinase activity and the transcriptional activation of HNF1␣ by Mirk, suggesting that Mirk, like p38, is activated by certain environmental stress agents. The Mirk-binding protein DCoH has been shown to be selectively expressed in colon carcinomas but not in normal tissue. Mirk may function as an HNF1␣ transcriptional activator in response to an MKK3-mediated stress signal, and the selective expression of DCoH could restrict the Mirk response to carcinoma cells. Mirk1 /Dyrk1B is a serine/threonine protein kinase that is expressed at elevated levels in normal skeletal muscle and certain carcinoma cell lines and at low levels in many normal tissues (1). Colon carcinoma cell lines stably overexpressing Mirk proliferated in serum-free medium (1), but the mechanism of Mirk action that enabled this survival capacity is unknown. Mirk is a member of the Dyrk/minibrain family of dual specificity, tyrosine-regulated, arginine-directed protein kinases (2-4) and is identical to Dyrk1B (5). Mirk/Dyrk1B and the related kinase Dyrk1A exhibit 54% amino acid identity with 90% identity or homology within the conserved kinase domain. Several lines of evidence indicate that Dyrk1A mediates neuronal differentiation. Dyrk1A has been mapped to the Down's syndrome critical region of chromosome 21, overexpression of Dyrk1A has been found in the Down's syndrome fetal brain (6), and transgenic mice overexpressing Dyrk1A exhibit cognitive deficits and motor abnormalities characteristic of Down's syndrome (7). Dyrk1A has been shown to phosphorylate the cAMP-response element-binding protein (CREB) in vivo, leading to the stimulation of subsequent cAMP response element-mediated ...
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